Process for prostaglandin intermediate

ABSTRACT

EPOXIDATION OF PROSTAGLANDIN A2 COMPOUNDS WITH HYDROGEN PEROXIDE OR AN ORGANIC TERTIARY HYDROPEROXIDE IN THE PRESENCE OF A BASE CONSISTING OF LITHIUM HYDROXIDE, OR A LITHIUM OR MAGNESIUM ALKOXIDE IN A TEMPERATURE RANGE OF -60*C. TO -10*C. YIELD AN INTERMEDIATE USEFUL FOR THE PREPARATION OF PROSTAGLANDIN E2 COMPOUNDS. THE PROSTAGLANDIN PRODUCTS HAVE PHARMACOLOGICAL UTILITY.

United States Patent Oflice 3,745,178 Patented July 10, 1973 3,745,178PROCESS FOR PROSTAGLANDIN INTERMEDIATE David R. White, Kalamazoo, Mich.,assignor to The Upjohn Company, Kalamazoo, Mich. No Drawing. Filed July2, 1971, Ser. No. 159,469 Int. Cl. C0711 1/20, 1/22 US. Cl. 260-3485 L 7Claims ABSTRACT OF THE DISCLOSURE Epoxidation of prostaglandin Acompounds with hydrogen peroxide or an organic tertiary hydroperoxide inthe presence of a base consisting of lithium hydroxide, or a lithium ormagnesium alkoxide in a temperature range of -60 C. to C. yields anintermediate useful for the preparation of prostaglandin E compounds.The prostaglandin products have pharmacological utility.

DESCRIPTION 'OF THE INVENTION 6 4 2 CODE 14 1s 1s 20 (I) Some of thederivatives of prostanoic acid are known as prostaglandins. One ofthose, prostaglandin E (PGE has the following formula:

Another, prostaglandin F (PGF has the formula:

I W o H H on m Still another, prostaglandin F (PGF- has the formula:

In Formulas I. to IV and in the formulas recited hereinafter in thespecification and claims, broken line attachments to the cyclopentanering indicate substituents in alpha configuration, i.e., below the planeof the cyclopentane ring. Heavy solid line attachments to thecyclopentane ring indicate substituents in beta configuration, i.e.,above the plane of the cyclopentane ring.

The side-chain hydroxy at C-15 in Formulas II to IV is in S (alpha)configuration. That configuration is shown by attachment of saidside-chain hydroxy to C-15 with a dotted line and hydrogen with a heavysolid line. The alternative configuration for the side-chain hydroxy atC-15 is known as R or epi (beta), and is shown when necessary byattachment of said side-chain hydroxy to C-15 with a heavy solid lineand hydrogen with a dotted line, thus The prostaglandin corresponding toPGE (Formula II) but with the R or epi configuration at 0-15 will bedesignated l5flPGE2. See Nature, 212, 38 (1966) for discussion of thestereochemistry of the prostaglandins.

These conventions regarding formulas, names, and symbols for derivativesof prostanoic acid apply to the formulas, names, and symbols givenhereinafter in the specification and claims. When reference is madehereinafter to the compounds of Formulas II to IV, by the symbols PGEPGF 0r PGF or to the methyl esters of any of those, 15(8) configurationwill be intended and by established custom, S or alpha will not bementioned in the name or symbol. For all of the other compounds recitedhereinafter, the configuration at C-15 will be identified in the name as1549 whenever the 15(R) configuration is intended.

Molecules of the known prostaglandins each have several centers ofasymmetry, and can exist in racemic (optically inactive) form and ineither of the two enantiomeric (optically active) forms, i.e., thedextrorotatory and levorotatory forms. As drawn, Formulas II to IV eachrepresent the particular optically active form of the prostaglandinwhich is obtained from certain mammalian tissues, for example, sheepvesicular glands, swine lung, or human seminal plasma, or by carbonyland/or double bond reduction of a prostaglandin so obtained. See, forexample, Bergstrom et al., Pharmacol. Rev. 20, l (1968) and referencescited therein.

The several aspects of this invention relate to novel methods forpreparing PGE PGFza, and PGF their acetates and methyl esters, and theISfi-epimers of those compounds, to novel intermediates used in thosemethods, to novel methods used to make those intermediates, and tocertain novel and pharmacologically useful analogs of PGE PG'F and PGFThe novel and pharmacologically useful PGE PGF and PGF analogs of thisinvention have the formulas:

no u on v11 In Formulas V, VI, and VII, R is hydrogen, alkyl of one to 8carbon atoms, inclusive, cycloalkyl of 3 to 10 carbon atoms, inclusive,aralkyl of 7 to 12 carbon atoms, in elusive, phenyl, or phenylsubstituted with one to 3 chloro or alkyl of one to 4 carbon atoms,inclusive. Also encompassed by Formulas V, VI, and VII arepharmacologically acceptable salts when R, is hydrogen. In Formulas Vand VII, Y is i H\ [011 or H, on In Formula V, B is r H OH In FormulaVI, indicates attachment to the ring in alpha or beta configuration.

It will be observed that each of the novel compounds of Formulas V andVI has a hydroxy group attached to the ll-position in betaconfiguration. In PGEg, PGF and PGF and in the compounds of Formula VII,the hydroxy at OH is attached in alpha configuration.

. With regard to Formulas V, VI, and VII, examples of alkyl of one to 8carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, and isomeric forms thereof. Examples of cycloalkylof 3 to 10 carbon atoms, inclusive, which includes alkyl-substitutedcycloalkyl, are cyclopropyl, Z-methylcyclopropyl,2,2-dimethylcyclopropyl, 2,3-diethylcyclopropyl, Z-butylcyclopropyl,cyclobutyl, Z-methylcyclobutyl, 3-propylcyclobutyl,2,3,4-triethylcyclobutyl, cyclopentyl, 2,2-dimethylcyclopentyl,3-pentylcyclopentyl, 3-tert-butylcyclopentyl, cyclohexyl,4-tert-butylcyclohexyl, 3-isopropylcyclohexyl, 2,2-dimethylcyclohexyl,cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Examples of aralkylof 7 to 12 carbon atoms, inclusive, are benzyl, phenethyl,l-phenylethyl, 2-phenylpropyl, 4-phenylbutyl, 3-phenylbutyl, 2-(1-naphthylethyl), and I-(Z-naphthylmethyl). Examples of phenyl substitutedby one to 3 chloro or alkyl of one to 4 carbon atoms, inclusive, arep-chlorophenyl, m-chlorophenyl, o-chlorophenyl, 2,4-dichlorophenyl,2,4,6-trichlorophenyl, p-tolyl, m-tolyl, o-tolyl, p-ethylphenyl,p-tertbutylphenyl, 2,5-dimethylphenyl, 4-chloro-2-methylphenyl, and2,4-dichloro-3-methylphenyl.

The known prostanoic acid derivatives, PGE PGF and PGF and their estersand pharmacologically acceptable salts are extremely potent in causingvarious biological responses. For that reason, these compounds areuseful for pharmacological purposes. See, for example, Bergstrom et al.,cited above, and references cited therein. A few of those biologicalresponses are systemic arterial blood pressure lowering in the case ofthe PGE and PGE compounds as measured, for example, in anesthetized(pentobarbital sodium) pentolinium-treated rats with indwelling aorticand right heart cannulas; pressor activity, similarly measured, for thePGF compounds; stimulation of smooth muscle as shown, for example, bytests on strips of guinea pig ileum, rabbit duodenum, or gerbil colon;potentiation of other smooth muscle stimulants; antilipolytic activityas shown by antagonism of epinephrine-induced mobilization of free fattyacids or inhibition of the spontaneous release of glycerol from isolatedrat fat pads; inhibition of gastric secretion in the case of the PGEcompounds as shown in dogs with secretion stimulated by food orhistamine infusion; activity on the central nervous system; controllingspasm and facilitating breathing in asthmatic conditions; de-

crease of blood platelet adhesiveness as shown by plateletto-glassadhesiveness, and inhibition of blood platelet aggregation and thrombusformation induced by various physical stimuli, e.g., arterial injury,and various biochemical stimuli, e.g., ADP, ATP, serotonin, thrombin,and collagen; and in the case of the PGE compounds, stimulation ofepidermal proliferation and keratinization as shown when applied inculture to embryonic chick and rat skin segments.

Because of these biological responses, these known prostaglandins areuseful to study, prevent, control, or alleviate a wide variety ofdiseases and undesirable physiological conditions in birds and mammals,including humans, useful domestic animals, pets, and zoologicalspecimens, and in laboratory animals, for example, mice, rats, rabbits,and monkeys.

For example, these compounds, and especially the PGE compounds, areuseful in mammals, including man, as nasal decongestants. For thispurpose, the compounds are used in a dose range of about 10 ,ug. toabout 10 mg. per ml. of a pharmacologically suitable liquid vehicle oras an aerosol spray, both for topical application.

The PGE and PGE, compounds are useful in the treatment of asthma. Forexample, these compounds are useful as bronchodilators or as inhibitorsof mediators, such as SRS-A and Histamine which are released from cellsactivated by an antigen-antibody complex. Thus, these compounds controlspasm and facilitate breathing in conditions such as bronchial asthma,bronchitis, bronchiectasis, pneumonia and emphysema. For these purposes,these compounds are administered in a variety of dosage forms, e.g.,orally in the form of tablets, capsules, or liquids; rectally in theform of tablets, capsules, or liquids; rectally in the form ofsuppositories; parenterally, subcutaneously, or intramuscularly, withintravenous administration being preferred in emergency situations; byinhalation in the form of aerosols or solutions for nebulizers; or byinsufflation in the form of powder. Doses in the range of about 0.01 to5 mg. per kg. of body weight are used 1 to 4 times a day, the exact dosedepending on the age, weight, and condition of the patient and on thefrequency and route of administration. For the above use theseprostaglandins can be combined advantageously with other antiasthmaticagents, such as sympathomimetics (isoproterenol, phenylephrine,ephedrine, etc.); xanthine derivatives (theophylline and aminophyllin);and corticosteroids (ACTH and prednisolone). Regarding use of these compounds see South African Pat. No. 681,055. The PGE compounds are usefulin mammals, includmg man and certain useful animals, e.g., dogs andpigs, to reduce and control excessive gastric secretion, therebyreducing or avoiding gastrointestinal ulcer formation, and acceleratingthe healing of such ulcers already present in the gastrointestinaltract. For this purpose, the compounds are injected or infusedintravenously, subcutaneously, or mtramuscularly in an infusion doserange about 0.1 pg. to about 500 ng. per kg. of body weight per minute,or in a total daily dose by injection or infusion in the range about 0.1to about 20 mg. per kg. of body weight per day, the exact dose dependingon the age, weight, and condition of the patient or animal, and on thefrequency and route of administration.

The PGE PGF,,, and PGE, compounds are useful whenever it is desired toinhibit platelet aggregation, to reduce the adhesive character ofplatelets, and to remove er prevent the formation of thrombi in mammals,includmg man, rabbibts, and rats. For example, these compounds areuseful in the treatment and prevention of myocardial infarcts, to treatand prevent post-operative thrombosis, to promote potency of vasculargrafts following surgery, and to treat conditions such asatherosclerosis, arteriosclerosis, blood clotting defects due tolipemia, and other clinical conditions in which the underlying etiologyis associated with lipid imbalance or hyperlipidemia. For thesepurposes, these compounds are administered systemically, e.g.,intravenously, subcutaneously, intramuscularly, and in the form ofsterile implants for prolonged action. For rapid response, especially inemergency situations, the intravenous route of administration ispreferred. Doses in the range about 0.005 to about 20 mg. per kg. ofbody weight per day are used, the exact dose depending on the age,weight, and condition of the patient or animal, and on the frequency androute of administration.

The PGE PGE and PGE compounds are especially useful as additives toblood, blood products, blood substitutes, and other fluids which areused in artificial extracorporeal circulation and perfusion of isolatedbody portions, e.g., limbs and organs, whether attached to the originalbody, detached and being preserved or prepared for transplant, orattached to a new body. During these circulations and perfusions,aggregated platelets tend to block the blood vessels and portions of thecirculation apparatus. This blocking is avoided by the presence of thesecompounds. For this purpose, the compound is added gradually or insingle or multiple portions to the circulating blood, to the blood ofthe donor animal, to the perfused body portion, attached or detached, tothe recipient, or to two or all of those at a total steady state dose ofabout 0.001 to mg. per liter of circulating fluid. It is especiallyuseful to use these compounds in laboratory animals, e.g., cats, dogs,rabbits, monkeys, and rats, for these purposes in order to develop newmethods and techniques for organ and limb transplants.

The PGE compounds are extremely potent in causing stimulation of smoothmuscle, and are also highly active in potentiating other known smoothmuscle stimulators, for example, oxytocic agents, e.g., oxytocin, andthe various ergot alkaloids including derivatives and analogs thereof.Therefore PGE for example, is useful in place of or in combination withless than usual amounts of these known smooth muscle stimulators, forexample, to relieve the symptoms of paralytic ileus, or to control orprevent atonic uterine bleeding after abortion or delivery, to aid inexpulsion of the placenta, and during the puerperium. For the latterpurpose, the PGE- compound is administered by intravenous infusionimmediately after abortion or delivery at a dose in the range about 0.01to about 50 ng. per kg. of body weight per minute until the desiredeffect is obtained. Subsequent doses are given by intravenous,subcutaneous, or intramuscular injection or infusion during puerperiumin the range 0.01 to 2 mg. per kg. of body weight per day, the exactdose depending on the age, weight and condition of the patient oranimal.

The PGE and PGF compounds are useful as hypotensive agents to reduceblood pressure in mammals, including man. For this purpose, thecompounds are administered by intravenous infusion at the rate about0.01 to about 50 g. per kg. of body weight per minute, or in single ormultiple doses of about 25 to 500 ,ug. per kg. of body weight total perday.

The PGE PGA and PGF compounds also increase the flow of blood in themammalian kidney, thereby increasing volume and electrolyte content ofthe urine. Therefore, these compounds are useful in managing cases ofrenal disfunction, especially those involving blockage of the renalvascular bed. Illustratively, the compounds are useful to alleviate andcorrect cases of edema resulting, for example, from massive surfaceburns, and in the management of shock. For these purposes, the compoundsare preferably first administered by intravenous injection at?! dose inthe range 10 to 1000 ,ug. per kg. of body weight or by intravenousinfusion at a dose in the range 0.1 to rg. per kg. of body weight perminute until the desired effect is obtained. Subsequent doses are givenby intravenous, intramuscular, or subcutaneous injection or infusion inthe range 0.05 to 2 mg. per kg. of body weight per day.

The PGE PGF and PGF- compounds are useful in place of oxytocin to inducelabor in pregnant female animals, including man, cows, sheep, and pigs,at or near term, or in pregnant animals with intrauterine death of thefetus from about 20 weeks to term. For this purpose, the compound isinfused intravenously at a dose 0.01 to 50 ,ug. per kg. of body weightper minute until or near the termination of the second stage of labor,i.e., expulsion of the fetus. These compounds are especially useful whenthe female is one or more weeks post-mature and natural labor has notstarted, or 12 to 60 hours after the membranes have ruptured and naturallabor has not yct started.

The PGF PGF and PGE compounds are useful for controlling thereproductive cycle in ovulating female mammals, including humans andanimals such as monkeys, rats, rabbits, dogs, cattle, and the like. Forthat purpose, PGE or PGF for example, is administered systematically,e.g., intravenously, subcutaneously, and intravaginally, at a doselevelin the range 0.001 mg. to about 20 mg. per kg. of body weight ofthe female mammal, ad-

vantageously during a span of time starting approximately at the time ofovulation and ending approximately at the next expected time of mensesof just prior to that time. Additionally, expulsion of an embryo orfetus (abortion) is accomplished by similar administration of thecompound during the first third of the normal mammalian gestationperiod.

As mentioned above, the PGE compounds are potent antagonists ofepinephrine-induced mobilization of free fatty acids. For this reason,this compound is useful in experimental medicine for both in vitro andin vivo studies in mammals, including man, rabbits, and rats, intendedto to lead to the understanding, prevention, symptom alleviation, andcure of diseases involving abnormal lipid mobilization and high freefatty acid levels, e.g., diabetes mellitus, vascular diseases, andhyperthyroidism.

The novel Formula-V, -VI, and -VII PGE PGF and PGF analogs of thisinvention each cause the biological responses described above for PGEPGF and PGF respectively, and each of these novel compounds isaccordingly useful for the above-described corresponding purposes, andis used for those purposes in the same manner as described above.

PGE PGF and PGF and their esters and pharmacologically acceptable saltsare all potent in causing multiple biological responses even at lowdoses. For example, PGE is extremely potent in causing vasodepressionand smooth muscle stimulation, and is also potent as an antilipolyticagent. Moreover, for many applications, these known prostaglandins havean inconveniently short duration of biological activity. In strikingcontrast, the novel analogs of Formulas V, VI, and VII are substantiallymore specific with regard to potency in causing prostaglandinlikebiological responses, and have a substantially longer duration ofbiological activity. Therefore, each of these novel prostaglandinanalogs is surprisingly and unexpectedly more useful than one of thecorresponding abovementioned known prostaglandins for at least one ofthe pharmacological purposes because it has a different and narrowerspectrum of biological activity than the known prostaglandins, andtherefore is more specific in its activity and causes smaller and fewerundesired side effects than when the known prostaglandin is used for thesame purpose. Moreover, because of its prolonged activity, fewer andsmaller doses of the novel prostaglandin analog can frequently be usedto attain the desired result.

The novel Formula-V, -VI, and -VII prostaglandin analogs are used asdescribed above in free acid form in ester form, or in pharmacologicallyacceptable salt form. When the ester form is used, the alkyl esters arepreferred, especially the alkyl esters wherein the alkyl moiety containsone to 4 carbon atoms, inclusive. Of those alkyl, methyl and ethyl areespecially preferred for optimum absorption of the compound by the bodyor experimental animal system.

Pharmacologically acceptable salts of these prostaglandin analogs usefulfor the purposes described above are those with pharmacologicallyacceptable metal cations, ammonium, amine cations, or quaternaryammonium cattons.

Especially preferred metal cations are those derived from the alkalimetals, e.g., lithium, sodium and potassium, and [from the alkalineearth metals, e.g., magnesium and calcium, although cationic forms ofother metals, e.g., aluminum, zinc and iron, are within the scope ofthis invention.

Pharmacologically acceptable amine cations are those derived fromprimary, secondary, or tertiary amines. Examples of suitable amines aremethylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine,triiso propylamine, N-methylhexylamine, decylamine, dodecylamine,allylamine, crotylamine, cyclopentylamine, dicyclohexylamine,benzylamine, dibenzylamine, a-phenylethylamine, fi-phenylethylamine,ethylenediamine, diethylenetriamine, and like aliphatic, cycloaliphatic,and araliphatic amines containing up to and including about 18 carbonatoms, as well as heterocyclic amines, e.g., piperidine, morpholine,pyrrolidine, piperazine, and lower-alkyl derivatives thereof, e.g., 1methylpiperidine, 4 ethylmorpholine, l-isopropylpyrrolidine,Z-methylpyrrolidine, 1,4- dimethylpiperazine, Z-methylpiperidine, andthe like, as well as amines containing water-solubilizing or hydrophilicgroups, e.g., mono-, di-, and triethanolamine, ethyldiethanolamine,N-butylethanolamine, Z-amino-l-butanol, 2-amino-2-ethyl-1,3-propanediol,2-amino-2 methyl 1- propanol, tris(hydroxyrnethyl)aminomethane,N-phenylethanolamine, N-(p-tert-arnylphenyl)diethanolamine, galactamine,N methylglucamine, N methylglucosamine, ephedrine, phenylephrine,epinephrine, procaine, and the like.

Examples of suitable phanmacologic-ally acceptable quaternary ammoniumcations are tetramethylammonium, tetraethylammonium,benzyltrimethylammonium, phenyltriethylammonium, and the like.

As discussed above, these novel prostaglandin analogs are administeredin various ways for various purposes; e.g., intravenously,intramuscularly, subcutaneously, orally, intra-vaginally, rectallyl,buccally, sublingually, topically, and in the form of sterile implantsfor prolonged action.

For intravenous injection or infusion, sterile aqueous isotonicsolutions are preferred. For that purpose, it is preferred because ofincreased water solubility to use the free acid form or thepharmacologically acceptable salt form. For subcutaneous orintramuscular injection, sterile solutions or suspensions of the acid,salt, or ester form in aqueous or non-aqueous media are used. Tablets,capsules, and liquid preparations such as syrups, elixirs, and simplesolutions, with the usual pharmaceutical carriers are used for oral orsublingual administration. For rectal or vaginal administration,suppositories, tampons, ring devices, and preparations adapted togenerate sprays or foams or to be used for lavage, all prepared as knownin the art, are used. For tissue implants, a sterile tablet or siliconerubber capsule or other object containing or impregnated with thesubstance is used.

The novel compounds of Formulas V, VI, and VII wherein R is other thanhydrogen, i.e., the esters wherein R is alkyl of one to 8 carbon atoms,inclusive, cycloalkyl of 3 to 10 carbon atoms, inclusive, aralkyl of 7to 12 carbon atoms, inclusive, phenyl, or phenyl substituted with one to3 chloro or alkyl of one to 4 carbon atoms, inclusive, are prepared fromthe corresponding acids of Formulas V, VI, and VII, i.e., wherein R ishydrogen, by methods known in the art. For example, the alkyl,cycloalkyl, and aralkyl esters are prepared by interaction of said acidswith the appropriate diazohydrocarbon. For example, when diazomethane isused, the methyl esters are produced. Similar use of diazoethane,diazobutane, 1- diazo-Z-ethylhexane, diazocyclohexane, andphenyldiazomethane, for example, gives the ethyl, butyl, 2-ethylhexyl,cyclohexyl, and benzyl esters, respectively.

Esterification with diazohydrocarbons is carried out by mixing asolution of the diazohydrocarbon in a suitable inert solvent, preferablydiethyl ether, with the acid reactant, advantageously in the same or adifferent inert diluent. After the esterification reaction is complete,the solvent is removed by evaporation, and the ester purified if desiredby conventional methods, preferably by chromatography. It is preferredthat contact of the acid reactants with the diazohydrocarbon be nolonger than necessary to effect the desired esterification, preferablyabout one to about ten minutes, to avoid undesired molecular changes.Diazohydrocarbons are known in the art or can be prepared by methodsknown in the art. See, for example, Organic Reactions, John Wiley &Sons, Inc., New York, N.Y., vol. 8, pp. 389394 (1954).

An alternative method for esterification of the carboxyl moiety of thenovel PGF-type or PGE-type compounds of Formulas V, VI, and VIIcomprises transformation of the free acid to the corresponding silversalt, followed by interaction of that salt with an alkyl iodide.Examples of suitable iodides are methyl iodide, ethyl iodide, butyliodide, isobutyl iodide, tert-butyl iodide, cyclopr-opyl iodide,cyclopentyl iodide, benzyl iodide, pheneth'yl iodide, and the like. Thesilver salts are prepared by conventional methods, for example, bydissolving the acid in cold dilute aqueous ammonia, evaporating theexcess ammonia at reduced pressure, and then adding the stoichiometricamount of silver nitrate.

The phenyl and substituted phenyl esters of the FormulaV, -IV, and -VIIcompounds are prepared by silylating the acid to protect the hydroxygroups, for example, replacing each OH with -O--Si(CH Doing that mayalso change -COOH to COOSi-(CH A brief treatment of the silylatedcompound with water will change COOSi-(CH back to -COOH. Procedures forthis silylation are known in the art and are discussed hereinafter.Then, treatment of the silylated compound with oxalyl chloride gives theacid chloride which is reacted with phenol or the appropriatesubstituted phenol to give a silylated phenyl or substituted phenylester. Then the silyl groups, e.g., OSi(CH are changed back to --OH bytreatment with dilute acetic acid. Procedures for these transformationsare known in the art.

The novel Formula-V, -VI, and -VII acids (R is hydrogen) are transformedto pharmacologically acceptable salts by neutralization with appropriateamounts of the corresponding inorganic or organic base, examples ofwhich correspond to the cations and amines listed above. Thesetransformations are carried out by a variety of procedures known in theart to be generally useful for the preparation of inorganic, i.e., metalor ammonium salts, amine acid addition salts, and quaternary ammoniumsalts. The choice of procedure depends in part upon the solubilitycharacteristics of the particular salt to be prepared. In the case ofthe inorganic salts, it is usually suitable to dissolve the acid inwater containing the stoichiometric amount of a hydroxide, carbonate, orbicarbonate corresponding to the inorganic salt desired. For example,such use of sodium hydroxide, sodium carbonate, or sodium bicarbonategives a solution of the sodium salt of the prostanoic acid derivative.Evaporation of the water or addition of a watermiscible solvent ofmoderate polarity, for example, a lower alkanol or a lower alkanone,gives the solid inorganic salt if that form is desired.

To produce an amine salt, the acid is dissolved in a suitable solvent ofeither moderate or low polarity. Examples of the former are ethanol,acetone, and ethyl acetate. Examples of the latter are diethyl ether andbenzene. At least a stoichiometric amount of the amine corresponding tothe desired cation is then added to that solution. It the resulting saltdoes not precipitate, it is usually 9 obtained in solid form by additionof a miscible diluent of low polarity or by evaporation. If the amine isrelatively volatile, any excess can easily be removed by evapo ration.It is preferred to use stoichiometric amounts of the less volatileamines.

Salts wherein the cation is quaternary ammonium are produced by mixingthe acid with the stoichiometric amount of the corresponding quaternaryammonium hydroxide in water solution, followed by evaporation of thewater.

The novel compounds of Formulas V, VI, and VII wherein R is hydrogen ormethyl, i.e., the free acids and the methyl esters, and also PGE PGF andPGF and the methyl esters of those are prepared by novel methods whichare described hereinafter. For those methods, one of the followingstarting materials is used:

In Formulas VIIa, VIII, and IX, R is either hydrogen or methyl, and R ishydrogen or acetyl.

It will be observed that the compounds encompassed by Formula VIIa arealso encompassed by VII. Thus, some Formula-VII compounds are usefulboth as intermediates and for pharmacological purposes.

These Formula-VIIa, -VIII, and -IX starting materials are allderivatives of prostanoic acid. The compounds of Formula VIII are knownin the art or are available by methods known in the art. See, forexample, Weinheimer et al., Tetrahedron Letters, No. 59, 5185 (1969); H.W. Youngken, Jr. (ed), Food-Drugs from the Sea," Proc. Marine TechnologySociety, pp. 311-314 (1969). The Formula-VIII compound wherein R and Rare both hydrogen is designated 1S 3-PGA alternatively 15(R)- PGA or15-epi-PGA The other compounds encompassed by Formula VHI are designatedISfi-PGA acetate, ISB-PGA methyl ester, and 15fi-PGA acetate methylester.

The compounds of Formula VIIa are new in the art and methods forpreparing them are described hereinafter. The Formula-VIIa compoundwherein R and R are both hydrogen is dsignated 15 3-PGE alternatively 15(R) PGE or IS-epi-PGE The other compounds encompassed by Formula VIIaare designated ISfi-PGE- 15-acetate, 15,8-PGE methyl ester, and ISfi-PGEacetate methyl ester.

The compounds of Formula IX are known in the art. See, for example,British specification 1,097,533. Novel methods for preparing theseFormula-IX compounds are described hereinafter. The Formula-IX compoundwherein R and R are hydrogen is designated PGA The other compoundsencompassed by Formula IX are designated PGA acetate, PGA methyl ester,and PGA acetate methyl ester.

All of the compounds of Formulas VIIa, VIII, and IX are obtained byextraction from a marine invertebrate. The compounds of Formula VIIa andVIII, i.e., the 15,8 compounds, are obtained from colonies of Plexaurahomomalla (Esper), 1792, forma R. The compounds of Formula IX, i.e., the15(8) of alpha compounds, are obtained from colonies of Plexaurahomomalla (Esper), 1792, forma S.

These Plexaura homomalla forms are members of the subclass Octocorallia,order Gorgonacea, suborder Holaxonia, family Plexauridae, genusPlexaura. See, for example, Bayer, The Shallow-Water Octocorallia of theWest Indian Region, Martinus Nijhoff, The Hague (1961). Colonies ofthese Plexaura homomalla forms are abundant on the ocean reefs in thezone from the lowtide line to about 25 fathoms in the tropical andsubtropical regions of the Western part of the Atlantic Ocean, fromBermuda to the reefs of Brazil, including the eastern shore reefs ofFlorida, the Caribbean island and mainland reefs, and the Gulf of Mexicoisland and mainland reefs. These colonies are bush-like or smalltree-like in habit, and are readily identified for collection asPlexaura homomalla (Esper), 1792, by those of ordinary skill in thisart. Forms R and S are distinguished by the methods described inPreparation 1 below.

The colonies of these two forms of Plexaura homomalla are easilyseparated into an outer bark-like cortex and an inner wiry proteinaceousstem or skeleton. Symbiotic algae or Zooxanthellae are also present inthe colonies. Weinheimer et al., cited above, disclose the occurence ofthe Formula-VIII compounds wherein R and R are both hydrogen and whereinR is methyl and R is acetyl in the air dried cortex of Plexaurahomomalla (Esper).

The choice of isolation or extraction method is determined by theparticular Formula-VIIa, -VIII, or -IX compound desired. Maximum yieldof the Formula-VIII or -IX diester is realized by freezing whole orcoarsely cut or chopped fresh Plexaura homomalla colonies within an hourand preferably sooner after the colonies are removed from the reef. Forsmall scale collections, this freezing is done advantageously bycontacting the colonies or pieces with solid carbon dioxide. For largerscale collections, other suitable freezing methods are known to the art.The frozen colonies or colony pieces should be kept frozen, preferablybelow about 20 C. until the extraction takes place.

The major component of fresh Plexaura homomalla (Esper), 1792, forma Ris ISfi-PGA acetate methyl ester, the Formula-VIII compound wherein R ismethyl and R is acetyl. Relatively minor components are the hydroxymethyl ester, the acetate, and the hydroxy acid of Formula VIII and theISB-PGE compounds encompassed by Formula VIIa. Of the latter, the15,8-PGE acetate methyl ester (R is methyl and R is acetyl) is the mostabundant. The major component of Plexaura homomalla (Esper), 1792, formaS is PGA acetate methyl ester, the Formula-IX compound wherein R ismethyl and R is acetyl. Relatively minor components are the hydroxymethyl ester, the acetate, and the hydroxy acid of Formula IX, and thePGE compounds corresponding to Formula VIIa but having the 15(8)configuration.

When the acetate methyl ester compound of Formula VIIa, VIII, or IX (Ris methyl, R is acetyl) is desired as a starting material, a suitablemethod comprises grinding the frozen whole Plexaura homomalla coloniesor colony pieces, advantageously in a hogger to a particle size with thelargest dimension about 5 mm., and then extracting the resultingparticles with any of the usual organic solvents, preferably one withmoderate to high polarity, e.g., dichloromethane or methanol,advantageously, for 15 to 30 minutes in a high speed mixer. The desiredcompounds are isolated from the extract by evaporation, and thenchromatography of the resulting residue. By this procedure, about 24 g.of 1SB-PGA acetate methyl ester, and about one g. each of ISB-PGA methylester and lSfi-PGE are obtained by dichloromethane extraction of 1500 g.of frozen Plexaura hamomalla (Esper), 1792, forma R colonies or colonypieces. Similarly, relatively large amounts of PGA acetate methyl esterare obtained from frozen Plexaura homomalla (Esper), 1792, forma S.

When the IS-hydroxy methyl ester of Formula VIIa, VIII, or IX (R ismethyl, R is hydrogen) is desired as a starting material, a suitablemethod comprises grinding the frozen whole Plexaura homomalla coloniesor colony pieces as above, and then contacting the resulting particleswith a lower alkanol, preferably methanol or ethanol, at 25 C. forseveral days. The solvent is then evaporated and the residuechromatographed to give substantially larger amounts of the hydroxymethyl ester compound relative to the acetate methyl ester compound.When the contact between the Plexaura homomalla particles and thealkanol is substantially shorter, substantially the same amount andratio of the various Formula- VIIa, -VII, or -IX compounds is obtainedwith the alkanol as with dichloromethane. An alternative method forobtaining these 15-hydroxy methyl esters is described below.

When l5fi-PGA ISBPGE or PGA (R and R in Formulas VIIa, VIII, and IX areboth hydrogen) are desired as starting materials in the novel processesof this invention, they are prepared from the corresponding methylesters and IS-acetate methyl esters after those have been extracted fromthe Plexaura homomalla colonies or colony pieces as described above. Asuitable method for removing the acetyl group of each of theFormula-VIIa, -VIII, and -IX IS-acetate methyl esters comprises mixingthe acetate methyl ester in lower alkanol solution, preferably inmethanol solution, with a strong acid, e.g., perchloric acid, for abouthours at 25 C. A suitable method for removing the methyl group of any ofthe Formula-VIIa, -VIII, and -IX methyl esters is the enzymatichydrolysis described in West Germany Offenlegungschrift No. 1,937,912,reprinted in Farmdoc Complete Specifications, Book No. 14, No. 6869R,Week R Mar. 18, 1970.

Another method for obtaining 15,B-PGA 15/3-PGE or PGA from Plexaurahomomalla colonies or colony pieces comprises freezing the Plexaurahomomalla colonies or colony pieces, preferably at a temperature belowabout C., and then allowing the colonies or colony pieces to thaw andwarm to a temperature in the range 20 to 30 C. The thawed colonies orcolony pieces are then maintained in the range 20 to 30 C. for at least24 hours. After that treatment, substantially none of the Formula-Vila,-VIII, and -IX compounds wherein R is methyl and R is acetyl arepresent, the principal Formula-Vila, -VIII and -IX compounds presentbeing those wherein R and R are both hydrogen, the minor componentsbeing those wherein R is methyl and R is hydrogen or wherein R ishydrogen and R is acetyl. As before, Formula-VIIa and -VIII compoundsare obtained from colonies of Plexaura homomalla (Esper), 1792, forma R,and Formula-IX compounds are obtained from colonies of Plexaurahomomalla (Esper), 1792, forma S.

A preferred procedure for the PGA and PGE type free acids comprisesgrinding the Plexaura homomalla colonies or colony pieces, preferably toa particle size with the largest dimension about 5 mm., and thenmaintaining the mixture in contact with water at a temperature in therange 20 to 30 C. for at least 24 hours. This mixture is filtered, andthe filtrate is extracted with an appropriate water-immiscible solvent,e.g., ethyl acetate. The solid residue is also extracted with anappropriate solvent, e.g., methanol. The two extracts are evaporated andthe total residue is chromatographed to give Formula- VIIa and -VIII orFormula-IX compounds, the principal 12 component in each case being thecompound wherein R; and R are both hydrogen.

Since our invention of the novel processes for transforming PGA and ISS-PGA and their methyl esters and acetate methyl esters to the variousprostanoic acids and esters disclosed herein, it has now been found thatsmall amounts of the 5,6-trans compounds of PGA; and 15B- PGA and theirmethyl esters and acetate methyl esters are also obtained from Plexaurahomomalla (Esper), 1792, forms R and S. These 5,6-trans compounds areextracted with and accompany the corresponding PGA type compoundsthrough many of their transformations. For example, PGA containing5,6-trans-PGA yields a mixture of PGE and 5,6-trans-PGE by the processrepresented in Chart E below.

\Vhen it is desired, for pharmacological purposes, to prepare the majorproducts of this invention free of 5,6- trans compounds, those 5,6-transcompounds are separated either from the starting reactants or from theproducts. In either case, several methods are available for separatingthe 5,6-trans-PG compounds from the PG com pounds. One method is bymeans of a silver-saturated ionexchange resin (for example, see E. A.Emken et al., J. Am. Oil Chemists Soc. 41, 388 (1964)), illustratedbelow in Preparations 5 and 6. The other method is by preferentiallyforming a mercuric acetate adduct of the 5,6-cis compound which isextractable into polar solvents illustrated below in Preparation 7.

Following the processes discussed herein and the procedures of theexamples below, the 5,6-trans-PG (and -15B-PG compounds are transformedto other 5,6-trans- P6 (and -l5fi-PG compounds, e.g., 5,6-trans-PGA to5,6-trans-PGE 5,6-trans-15 3-PGA acetate methyl ester to5,6-trans-15B-PGE acetate methyl ester, and the like.

As mentioned above, the Formula-VIIa, -VIII, and -IX compounds arestarting materials for the preparation of PGE PGF and PGF the methylesters of those, and also the novel compounds of Formulas V and VI, andsome of the novel compounds of Formula VII. The novel processes usingthese starting materials will now be described.

The Formula-VIII and -IX starting materials are both of the PGA-type.According to the novel processes of this invention, those are firsttransformed to corresponding PGE-type compounds. The chemical reactionsinvolved in those transformations are shown generically in Chart A.

In Chart A, R, is hydrogen, methyl, or --Si(A) wherein A is alkyl of oneto 4 carbon atoms, inclusive, aralkyl of 7 to 12 carbon atoms,inclusive, phenyl, or phenyl substituted with one or 2 fluoro, chloro,or alkyl of one to 4 carbon atoms, inclusive; G is H" 0R or H on.

wherein R is hydrogen, acetyl, or Si(A) when R, is hydrogen or methyl,and R is -Si(A) when R, is Si(A) R is hydrogen or methyl; and B is H" OHorH OH.

Thus, Formula X in Chart A encompasses the starting materials ofFormulas VIII and IX obtained from Plexaura homomalla, and alsocompounds of the formula:

wherein R is as defined above, and Z is wherein A is as defined above.

In Formula XI, of Chart A,

indicates attachment of the epoxy oxygen to the ring in alpha or betaconfiguration. In Formulas XII and XIII of Chart A, indicates attachmentof hydroxy to the ring in alpha or beta configuration.

CHART A C 0 R4 ii 1 (oxidation) 1 (reduction) 1 (hydrolysis) XIIcompounds with the 11 8 configuration of the novel Formula-V compoundsof this invention wherein Y is i.e., 1lB-PGE and llfl,l5B-PGE If theFormula-XII or -XIII product is to have the (S) configuration, e.g., PGEor 11,B-PGE then the Formula-X starting material should have the 15(S)configuration, i.e., G should be If a 156 compound of 'Formula XII orXHI is desired, e.g., lSB-PGE or llB,15,B-PGE then the Formula-X 14starting material should have the 15 (R) or 15-epi configuration, i.e.,G should be As described above, Formula-IX starting materials wherein Ris hydrogen or methyl and R is hydrogen, and with the 15(S)configuration, are obtained from Plexaura homomalla (Esper), 1792, formaS. Those same compounds are also produced by reacting the corresponding15 (R) (beta) compound with a hydrocarbyl or halohydrocarbyl sulfonylchloride or bromide, preferably a lower alkylsulfonyl chloride orbromide, especially methanesulfonyl chloride or bromide, or a benzeneorsubstituted-benzenesulfonyl chloride or bromide, e.g., ptoluenesulfonylchloride. This reaction is done in the presence of at least sufficienttertiary amine, e.g., triethylamine, to absorb the hydrogen chloride orhydrogen bromide lay-product, and at a low temperature, preferably inthe range -15 to +15 C. The presence of an inert liquid diluent, e.g.,tetrahydrofuran, is helpful to maintain a mobile homogenous reactionmixture. At 0C. and with methanesulfonyl chloride, usually 30 to 60minutes is a sufficient reaction time. The product is hydrolyzed to amixture of 15(S) (alpha) and 15(R) hydroxy compounds. These areseparated by procedures known in the art, and the 15 (S) product ispurified by procedures known in the art, advantageously bychromatography on silica gel. This reaction is also used to transform 15(S) Formula-IX starting materials wherein R is hydrogen or methyl and Ris hydrogen to the corresponding 15(R) compounds. In each case, amixture of 15(R) product and 15(S) starting material is obtained, thecomponents of which are separated as described above.

Another method of transforming a 15,6-PG compound to a PG compound is byconverting it to a mixture of PG 15-formate and ISB-PG 15-formatecompounds, separating the PG 15-formate, and hydrolyzing the PG 15-formate to the desired PG compound (see I. E. Pike et al., J. Org. Chem.34, 3552 (1969)).

The mixture of alpha and beta 15-formates is prepared by maintaining the158 compound, e.g. ISB-PGE 15,8-PGA or I5,B-PGF in formic acid buiferedwith an alkali metal formate in the range 10 to C. until a substantialamount of the 15,9 compound, e.g. 15,8-PGE 15-formate, has beentransformed to the PG 15-formate.

' The mixture of the PG 15-formate and 1513 15-formates thus obtained isthen separated by known methods, e.g. by chromatography.

The PG 15-formate can then be hydrolyzed to the desired PG l5-hydroxycompound. The ISfi-PG 15-formate yields the corresponding 15 3-PG15-hydroxy compound, which is then recycled through the above steps forfurther isomerization to the PG compound if desired.

This procedure is also useful to transform a PG compound to a 1519 PGcompound, by obtaining a mixture of the intermediate PG 15-formate and15fl-PG 15-formate compounds, separating them, and hydrolyzing them tothe respective PG 15-hydroxy and 15[3-PG 15-hydroxy compounds. In thiscase, the PG compound is recycled for further isomerization to the 15 8compound.

Referring again to Chart A, the transformation of starting material X toepoxide XI is carried out by reacting X with any agent known toepoxidize an a,}3-unsaturated ketone without reacting with isolatedcarbon-carbon double bonds, for example see Steroid Reactions, CarlDjerassi, ed., Holden-Day Inc., 1963, p. 593. Especially preferred areaqueous hydrogen peroxide or an organic tertiary hydroperoxide. See, forexample, Organic Peroxides, A. V. Tobolsky et al., IntersciencePublishers, N.Y., 1954. For this purpose, the peroxide or hydroperoxideis employed in an amount of at least one equivalent per mole ofFormula-X reactant in the presence of a strong base, e.g., an alkalimetal hydroxide, a metal alkoxide, or a quaternary ammonium hydroxide.For example, there is employed lithium hydroxide,

sodium hydroxide,

potassium hydroxide,

lithium ethoxide,

lithium octyloxide,

magnesium methoxide,

magnesium isopropoxide, benzyltrimethylammonium hydroxide,tetraethylammonium hydroxide, butyltrimethylammonium hydroxide,butyldiethylphenylammonium hydroxide, benzylethyldimethylammoniumhydroxide, benzyldimethyloctadecylammonium hydroxide,benzyldodecyldimethylammonium hydroxide, decyldimethylphenylammoniumhydroxide,

and the like. See, for example, Sidgwick, Organic Chemistry of Nitrogen,Third Edition, rev. by Miller and Springall, Oxford, 1966, pp. 116-127.

The ratio of alpha to beta epoxide formed in the reaction is related tofour factors: the epoxidizing agent, the base, the diluent, and thetemperature. Hydrogen peroxide is employed in the concentrations usuallyavailable, for example 3% to 90%, although 30% is especially convenient.When the alpha epoxide is the desired product, tert-butyl hydroperoxideis especially preferred as the epoxidizing agent. Examples of otherorganic tertiary hyroperoxides useful for this purpose are tert-pentylhydroperoxide, decahydronaphthyl hydroperoxide, agar-dimethylbenzylhydroperoxide, and 1,1-diphenylethyl hydroperoxide. The base is presentin the proportion of 0.1-3.0, preferably about 0.1-0.5 equivalent ofbase per mole of starting material X when R; is methyl and R is acetyl;preferably about 1.5-2.5 equivalent of base per mole of startingmaterial VIII or IX wherein R and R are hydrogen. When the alpha epoxideis the desired product, lithium hydroxide, lithium or magnesiumalkoxides of one to 8 carbon atoms, and benzyltrimethylammoniumhydroxide are the preferred bases, although the lithium and magnesiumcompounds are especially preferred.

It is advantageous to use an inert liquid diluent in the epoxidationstep to produce a mobile homogenous reaction mixture, for example, alower alkanol, dioxane, tetrahydrofuran, dimethoxyethane,dimethylsulfoxide, or dimethylsulfone. When the alpha epoxide ispreferred, tetrahydrofuran or the less polar dimethoxyethane areespecially preferred as the diluent. A reaction temperature in the range60 to 0 C. is generally preferred, especially below 10 C. The lowertemperatures below 30 C. are especially preferred for favoring formationof alph epoxide over beta epoxide. At a temperature of C., theepoxidation is usually complete in 3 to 6 hours. It is also preferredthat the reaction be carried out in an atmosphere of an inert gas, e.g.,nitrogen, helium, or argon. When the reaction is complete as shown bythe absence of starting material on TLC plates (3% acetone indichloromethane), the reaction mixture is neutralized, and the epoxyproduct is isolated by procedures known in the art, for example,evaporation of the diluent and extraction of the residue with anappropriate water-immiscible solvent, e.g., ethyl acetate.

This transformation of X to XI usually produces a mixture of Formula-XIalpha and beta epoxides both with either the 15(R) or 15(5)configuration depending on the configuration at 0-15 in the Formula-Xstarting material. Although these mixtures are separated into theindividual alpha and beta isomers, for example, by chromatography byprocedures known to be useful for separating alpha and beta epoxidemixtures, it is usually advantageous to transform the Formula-XI mixtureof alpha and beta epoxides to the corresponding mixture of Formula-XIIHot and H5 hydroxy compounds. The latter mixture is then readilyseparated into the Hot and 11 3 compounds, for example, bychromatography of silica gel.

During the transformation of epoxides XI toalcohols XII, an alphaepoxide yields an Hot-hydroxy compound, and a beta epoxide yields anIIB-hydroxy compound. The ratio of alpha to beta epoxides in XI, andhence the eventual ratio of Hot. and II alcohols in XII, produced fromstarting material X is dependent in large measure on the nature of R inX. Recall that G is defined as n ontorn 0R.

wherein R is hydrogen, acetyl, or Si--(A) wherein A is as defined above.For either definition of G, i.e., R configuration or S configuration,when R is hydrogen, more Formula-XI beta epoxide is formed than when Ris acetyl, and more Formula-XI beta epoxide is formed when R is acetylthan when R is -Si-(A) For example, when G in Formula X is H on,

the preferred basic hydrogen peroxide epoxidation gives about equalamounts of alpha and beta epoxides, but when G in Formula X is about 3parts of alpha epoxide and one part of beta epoxide are obtained, andwhen G in Formula X is about 4 parts of alpha epoxide and one part ofbeta epoxide are obtained, both reactions with the same epoxidationreagent. When G in Formula X is H, on,

about one part of alpha epoxide and 3 parts of beta epoxide are obtainedwith basic hydrogen peroxide, but when G is about 6 parts of alphaepoxide and 4 parts of beta epoxide are obtained with the sameepoxidation reagent.

Each of the novel Formula-V and -VI compounds of this invention has ahydroxy attached in beta configuration to the cyclopentane ring. Some ofthe compounds of Formula V, i.e., when Y is n" on or H On the otherhand, when a prostanoic acid product with the natural IIa configurationfor the hydroxy at 0-11 is the desired final product, e.g., PGE PGF orPFG there is advantage in choosing a Formula-X starting material whichgives a greater amount of the alpha epoxide H O-Si-UU:

or the corresponding 15 (S) compounds.

As mentioned above, the starting materials of Formula X encompass notonly the Formula-VIII and -IX compounds obtained from Plexaurahomoma'lla but also the silyl compounds of Formula Xa. When desired asreactants, these silyl compounds are prepared by silylation of PGA15/3-PGA or the methyl esters of those. These silylations are carriedout by procedures known in the art. See, for example, Pierce, Silylationof Organic Compounds, Pierce Chemical Co., Rockford, Ill. (1968). TheC-15 hydroxy group of PGA 15/3-PGA or their methyl esters is transformedto an --O-Si(A) moiety wherein A is as defined above, sufficientsilylating agent being used according to known procedures to accomplishthat. The necessary silylating agents for this purpose are known in theart or are prepared by methods known in the art. See, for example, Post,Silicones and Other Organic Silicon Compounds, Reinhold PublishingCorp., New York, N.Y. (.1949). In the case of PGA and l5fi-PGA excesssilylating agent and prolonged treatment also transform the COOH toCOO'Si-(A) It is optional in transforming X to XI whether or not thisCOOH of PGA or l5 8-PGA is esterified to COOSi (A) 3 since that estergroup is transformed to COOH during formation and isolation of theFormula-XI epoxide product.

The various As of a Si-(A) are alike or different. For example, a -Si(A)can be trimethylsilyl, dimethylphenylsilyl, or methylphenylbenzylsilyl.

When it is desired to retain the -Si(A) moiety at C-lS in the Formula-XIepoxide product, for example, to give steric control in a subsequentreaction, it is important in isolating the epoxide that the presence ofacid be avoided and that contact with water be minimized unless thewater is kept cold, i.e., below about 10 C.

Referring again to Chart A, the transformation of epoxide XI to hydroxycompound XII is accomplished by reduction with chromium (II) salts,e.g., chromium (II) chloride or chromium (H) acetate. Those salts areprepared by methods known in the art, e.g., Inorganic Syntheses, VIII,125 (1966); ibid., VI, 144 (1960); ibid. HI, 148 (1950); ibid. I, 122(1939); and references cited in those. This reduction is carried out byprocedures known in the art for using chromium (II) salts to reduceepoxides of one-unsaturated ketones to fi-hydroxy ketones. See, forexample, Cole et al., J. Org. Chem. 19, 131 (1954), and Neher et al.,Helv. Chem. Acta 42, 132 (1959). In these reactions, the absence of airand strong acids is desirable. If it is desired to maintain a -Si(A)moiety on 0-15, a neutral reaction mixture is preferred. An especiallypreferred procedure is to generate the chromium (II) ion in the presenceof the Formula-XI epoxide, for example, by mixing the epoxide with achromium (III) salt, e.g., the chloride, with metallic zinc in thepresence of acetic acid. The desired Formula-XII compound is isolatedfrom the reduction reaction mixture by methods known in the art, carebeing taken to minimize contact of the product with acid and water,especially warm water, when retention of a -Si(A) at C- is desired.

Unexpectedly, amalgamated aluminum metal has also been found to beuseful as a reducing agent in place of chromium (II) salts to transformFormula XI epoxides to Formula XII hydroxy compounds. This reagent waspreviously not known to be useful for this type of reaction. This use ofamalgamated aluminum represents a distinct and separate aspect of thisinvention.

Amalgamated aluminum is prepared by procedures known in the art, forexample, by contacting aluminum metal in the form of foil, thin sheet,turnings, or granules with a mercury (II) salt, for example, mercuricchloride, advantageously in the presence of sufficient water to dissolvethe mercury (II) salt. Preferably, the surface of the aluminum metal isfree of oxide. That is readily accomplished by physical removal of theusual oxide layer, e.g., by abrasion or scraping, or chemically, e.g.,by etching with aqueous sodium hydroxide solution. It is only necessarythat the aluminum surface be amalgamated. The amalgamated aluminumshould be freshly prepared, and maintained in the absence of air andmoisture until used.

The reductive opening of the Formula-XI epoxide ring is accomplished bycontacting said epoxide with the amalgamated aluminum in the presence ofa hydroxylic solvent and sufficient inert organic liquid diluent to givea mobile and homogeneous reaction mixture with respect to the hydroxylicsolvent and said epoxide. Among hydroxylic solvents, Water is especiallypreferred although lower alkanols, e.g., methanol and ethanols are alsooperable.

Examples of inert organic liquid diluents are normally liquid otherssuch as diethyl ether, tetrahydrofuran, dimethoxyethane, diglyme(dimethyl ether of diethylene glycol), and the like. Especiallypreferred is tetrahydrofuran. When a water-immiscible liquid diluent isused, a mixture of Water and methanol or ethanol is especially useful inthis reaction since the latter two solvents also aid in forming thedesired homogeneous reaction mixture. For example, a mixture of diethylether and water is used with suflicient methanol to give a homogeneousreaction mixture.

This reductive opening requires two hydrogen atoms per molecule ofepoxide. Amalgamated aluminum reacts readily with water and more slowlywith other hydroxylic solvents to give hydrogen. One atomic equivalentof aluminum required 3 molecular equivalents of the hydroxylic solventto give 3 atomic equivalents of hydrogen. Therefore, one molecularequivalent of epoxide requires two-thirds atomic equivalent of aluminumand two molecular equivalents of the hydroxylic solvent. Evolution ofhydrogen gas (H molecules) is observed during this reductive opening ofthe epoxide. It is not known whether the reductive opening is caused byhydrogen atoms or hydrogen molecules. However, some of the hydrogen gasescapes from the reaction mixture. Therefore, it is preferred to use anexcess of amalgamated aluminum and hydroxylic solvent, preferably atleast one atomic equivalent of aluminum and three molecular equivalentsof hydroxylic solvent per molecular equivalent of epoxide. Because ofthe relatively high economic value of the epoxide compared withamalgamated aluminum and hydroxylic solvents, it is preferred to assuremaximum yields of the desired Formula-XII hydroxy compound by use ofsubstantially greater excess of amalgamated aluminum and hydroxylicsolvent, e.g., up to ten times or more of those reagents than istheoretically required.

The reductive opening of the epoxide is carried out by mixing a solutionof the epoxide in the organic diluent with the amalgamated aluminum andthe hydroxylic solvent. Since the reaction is exothermic, it is usuallyadvantageous to cool the solution to a low temperature, e.g., 20 C. to 0C., before adding the amalgamated aluminum and hydroxylic solvent and tomaintain the reaction mixture in the range 20 to 30 C. by externalcooling. This is especially advantageous when water is used as thehydroxylic solvent. Higher reaction temperatures are operable but notpreferred when a high yield of the Formula-XII products'is desired.Stirring is preferred during the reaction since the reaction mixture isheterogeneous with respect to the solution and the amalgamated aluminum.

For reasons not understood, better yields and a shorter reaction timeare usually observed when only part of the amalgamated aluminum is addedat the start of the reac- H 000 OH:

in Formulas XI and XII, a suitable TLC system is ethylacetate-cyclohexane-acetic acid (40/60/2), the Formula- XI startingmaterial having r; 0.64, and the two Formula- XII products having r;0.25 (11;?) and r; 0.20 (Ha).

As a modification of the above-described process for reductive openingof the epoxide, it has been found quite unexpectedly that instead ofemploying a Formula-XI compound wherein R is hydrogen, the reductiveOpening reaction proceeds more smoothly and completely if there is used,instead, an epoxide of the formula wherein G and XIa are as definedabove and R is a cation of an alkali or alkaline earth metal or aquaternary ammonium group.

Thus, the Formula-XI epoxide compound is treated with a hydroxide oroxide of lithium, sodium, potassium, magnesium, calcium, barium, orstrontium prior to contacting with the aluminum amalgam. Optionally, thequaternary ammonium bases are used for this neutralization, for examplebenzyltrimethylammonium hydroxide. The base is used in equivalent amountto the acid so that R is replaced by the corresponding metal orquaternary ammonium cation. Alternatively, instead of the hydroxides oroxides, there are employed the hydrides, the carbonate, thebicarbonates, or the alkoxides, for example lithium hydride, potassiumcarbonate, sodium bicarbonate, magnesium methoxide, and the like, whichform the corresponding Formula-XIa salts with the Formula-XI free acid.Alternatively, a metal or quaternary-ammonium carboxylate compound orFormula-XIa salt carried forward from the epoxidation step, whetherisolated in that step or not, is employed in the reductive step withaluminum amalgam. It is preferred that the Formula-XIa salt be solublein the organic diluent-alkanol-water or organic diluent-water mediumused for the reduction step. By using the above-described salts, thereduction step proceeds smoothly without formation of insoluble aluminumsalts which hinder the reaction. Following the reduction or hydrolysisstep, the R cations are replaced with hydrogen by means known in theart, for example by acidification and extraction of the acid compoundinto an organic phase.

The desired Formula-XII hydroxy products are isolated by filtration ofthe reaction mixture, advantageously after addition of magnesium sulfateas a filter aid, and evaporation of the organic diluents. TheFormula-XII products are then hydrolyzed if desired to remove Si(A) from0-15, and the Ho: and 11p products of Formula XIII are separated, ifdesired, by procedures known in the art, e.g., chromatography on silicagel.

The products of Formula-XII are all of the PGE -type and include PGE PGEIS-acetate, PGE methyl ester, PGE IS-acetate methyl ester, PGE and PGEmethyl ester with a O-Si-(A) at C-l5, the corresponding 15;? compounds,and compounds corresponding to all of those wherein hydroxy is attachedto (3-11 in beta configuration.

As mentioned above, the transformation of X to XI to XII usually gives amixture of Formula-XII PGE-type products, part with alpha and part withbeta configura- 7 tion for the hydroxy at C-11. There are severalalternatives regarding that mixture. If -0Si(A) is attached to C-15,that can readily be transformed by hydrolysis to -OH. These hydrolysesare carried out by prior art procedures known to be useful fortransforming silyl ethers to alcohols. See, for example, Pierce, citedabove, especially p. 447 thereof. A mixture of water and sufficient of awater-miscible organic diluent to give a homo geneous hydrolysisreaction mixture represents a suitable reaction medium. Addition of acatalytic amount of an organic or inorganic acid hastens the hydrolysis.The length of time required for the hydrolysis is determined in part bythe hydrolysis temperature. With a mixture of water and methanol at 25C., several hours is usually suflicient for hydrolysis. At 0 C., severaldays is usually necessary. Also, if -OCOCH is attached to C-15, that canreadily be transformed to --OH by acid-catalyzed alcoholysis asdescribed above for removing the acetyl group of the Formula-VIH and -IXPGA-type starting materials. Both of those transformations are shown inChart A, i.e., XII to XIII. Either before or after those transformationsof XII to XIII, the F ormula-XII or -XIII mixture of 11a and H13 isomerscan be separated by methods known in the art, advantageously bychromatography on silica gel.

Further regarding the Formula-XIII compounds, those compounds whereinthe configuration of the hydroxy at 0-11 is beta are within the scope ofthe Formula-V novel compounds of this invention, and those compoundswherein the configuration of the hydroxy at C-11 is alpha and B is H onare within the scope of the Formula-VII novel compounds of thisinvention. Both groups of novel compounds are used for pharmacologicalpurposes described above for those compounds, the acids also beinguseful as reactants to prepare pharmacologically useful esters andpharmacologically acceptable and useful salts, both as described above.Moreover, Formula XIII compounds wherein the configuration of thehydroxy at C-ll is alpha and B is are PGE and 'PGE methyl ester, both ofknown pharmacological utility.

Still further regarding the separated compounds of Formulas XII andXIII, when a compound with one configuration at (3-11, either alpha orbeta, is desired as an intermediate or for pharmacological purposes, theother isomer is readily dehydrated to give additional Formula- XPGA-type starting material which is then used as a starting materialaccording to the processes defined in Chart A and procedures describedabove to give additional of the desired isomer. These dehydrations areaccomplished by procedures known in the art for dehydration of PGE-typecompounds to PGA-type compounds. See, for example, Pike et al., Proc.Nobel Symposium II, stockholm (1966), Interscience Publishers, New York,p. 162 (1967), and British specification 1,097,533. These are acidicdehydrations, and alkanoic acids of 2 to 6 carbon atoms, inclusive,especially acetic acid, are preferred for this purpose. Dilute aqueoussolutions of mineral acid, e.g., hydrochloric acid, especially in thepresence of a 21 solubilizing diluent, e.g., tetrahydrofuran are alsouseful as reagents for these acidic dehydrations, although thesereagents may also cause partial hydrolysis of the Formula-XII or XIIImethyl esters to carboxylic acids. A Si(A), moiety at C-15 is alsoremoved during all of these acidic dehydrations.

Still further regarding the Formula-XII and -XIII compounds, either asmixtures or separately, any of those is transformed to other usefulcompounds or mixtures by changing the PGE-type compounds to PGF-typeproducts by reducing the ring carbonyl at C-9 to alpha hydroxy or betahydroxy. Those transformations are shown in Chart B.

In Chart B, R, is hydrogen, methyl, or Si--(A) R is hydrogen or methyl,R is hydrogen or Si(A) and G is and indicates attachment to thecyclopentane ring in alpha or beta position.

The Chart B starting material XII is prepared as shown in Chart A. Thecompounds of Formula XIII in Chart A are included in Formula XII. Asdescribed above, 15fi-PGE CHART B XII R10 XIV R10 XV (hydrolysis oraleoholysls) no 3 xvr raphy.

22 15 8-PGE acetate, 15B-PGE methyl ester, and 15fi-PGE methyl esteracetate are obtained from Plexaura homvmalla (Esper), 1792, forma R. Allof these compounds are encompassed by Formula XII, and thus, extractionof this form of Plexaura homomalla provides an alternative source ofthese starting materials.

Referring to Chart B, the starting material XH can be a mixture ofcompounds with regard to the configuration of C-11, or the startingmaterial can be stereochemically pure with respect to 0-11, dependingupon whether there has been an earlier separation of 11a and 11B isomers(see above discussion of Chart A reactions).

The transformation of PGE-type starting material XII to PGF-type productXVI involves reduction of a ring carbonyl to a ring hydroxy. Thisprocess is known in the art for some of the compounds encompassed byFormula XII, i.e., when the configuration at C-ll is alpha and theconfiguration at 0-15 is S. For the other compounds encompassed byFormula XII, this reaction is novel, and novel Formula-XV and XVIcompounds are produced.

For this carbonylto-hydroxy reduction, methods known in the art areused. See, for example, Pike et al., J. Org. Chem. 34, 3552 (1969). Useis made of any of the known ketonic carbonyl reducing agents which donot reduce ester or acid groups or carbon-carbon double bonds. Examplesof those are the metal borohydrides, especially sodium, potassium,lithium, and zinc borohydrides, lithium (tri-tert-butoxy) aluminumhydride, metal trialkoxy borohydrides, e.g., sodiumtrimethoxyborohydride, and diisobutylaluminum hydride. The sodium,potassium, and zinc borohydrides are preferred for this reduction,especially zinc borohydride.

Unexpectedly, the amalgamated aluminum metal found useful above intransforming the Formula-XI epoxides to Formula-XII hydroxy compoundshas also been found use ful as an agent for this carbonyl-to-hydroxyreduction of PGE-type compounds to PGF-type compounds. Either thePGE-type salts or the PGE-type esters are employed, for example theFormula-XII hydroxy compounds produced from the Formula-XI epoxides withor without intermediate isolation. Furthermore, the Formula-XI epoxidesmay be subjected to the combined epoxide-reduction andcarbonyl-reduction reactions practically simultaneously by operating athigher temperatures, for example 40-60 0., although it is preferred forhigh yields of the ll-hydroxy compounds that the reductions be donestepwise. The solvents which are operable for this reduction aregenerally the same as those found useful for the epoxidereduction step.Somewhat higher temperatures or longer reaction times are required forthe carbonyl-to-hydroxy reduction, however. For example, at 25 0., about4 to 24 hours are required; at higher temperatures, e.g., 50-60" 0.,about one to 2 hours are suflicient.

This carbonyl reduction usually produces a mixture of PGF,,-type and PGF-type compounds, i.e., compounds with the alpha configuration andcompounds with the beta configuration for the hydroxy at 0-9. Thismixture of alpha, and beta isomers is separated by methods known in theart, e.g., chromatography on silica gel. See Pike et al., ibid., forexample. If the Formula-XII starting material isa mixture of 11a and 11Bisomers, then this reduction will usually produce four isomers, i.e.,9a,1lu, :,1113, 913,1lu, and 9fi,11,8. Those compounds are alsoseparated from such mixtures by silica gel chromatog- Regarding thetransformation of XII to XIV in Chart B,.it will be observed that theparameters for XII are such that all XII compounds are included in XIV.In other words, the transformation XII to XIV is an optional processstep in proceeding from XII to XV. The reason for this is as follows.During the reduction of XIV to XV, the ratio of 9'ot-hydroxy and9B-hydroxy compounds formed will be different when R; in XIV is hydrogenthan when R is Si(A) For example, with the Formula-XIV compound whereinR is hydrogen, G is H OH,

and R O- represents HO i.e., lla-hydroxy, sodium borohydride reductiongives 42 parts of the corresponding Formula-XV 9ot-hydroxy compound, and58 parts of the 9fl-hydroxy compound. But with the Formula-XIV compoundwherein R4 is hydrogen, G is H o-sionan,

and R O- represents (CH SiO sodium borohydride reduction gives 85 partsof the corresponding Formula-XV 9a-hydroxy compound and 15 parts of the9Bhydroxy compound. Similar differences are observed With the otherisomers encompassed by Formula XIV although not necessarily in the samedirection. Accordingly, whether R in Formula XIV is to be hydrogen orSi(A) depends on the particular Formula XV C-9 isomer desired and theinfluence of silylation on the isomer ratio. For any particularFormula-XIV starting material, the latter is readily determined by smallscale reduction with and without silylation. When silylation beforecarbonyl reduction is indicated, largely for economic reasons, it ispreferred that A be methyl, i.e., that R be This transformation of XIIto XIV wherein :R is Si-(A) is carried out as described above for thetransformation of hydroxy to O-Si(A) at C-15 prior to the Chart Areactions. When R is XII is hydrogen, the COOH is also transformed inpart or entirely to COOSi-(A) with prolonged silylation and excesssilylating agent. It is optional in transforming XII to XdV wherein R isSi(A) whether or not the COOH of XII is esterified to COO-Si(A) When Gin Formula X-IV is 11' OH or H on,

those OH are also transformed to OSi-(A) by this silylation.

With regard to the Formula-XV carbonyl reduction product (Chart B), whenthe method used to isolate said product does not remove any Si(A) groupswhich may be present, that is accomplished as described above for theremoval of -Si(A) groups from Formula-XII products (Chart A, XII toXIII). Also, when G in Formula XV is CHART C H OCOCHa or H oooom theacetyl is removed by alcoholysis also as described above for changingacetoxy at -15 to hydroxy. These reactions are shown in Chart B as XV toXVI.

When R in Formula XVI is methyl and the compound wherein R is hydrogenis desired, that methyl ester is saponified, by methods known in theart. See, for example, Just et al., I. Am. Chem. Soc. 91, 5371 (1969).This saponification also changes a C-15 acetate to a C-15 hydroxy.

The compounds encompassed by Formula XVII include the known compoundsPGF PFG and the methyl esters of those. Also included in Formula XVI arethe novel compounds 15,B-PFG 15,8-PFG and the methyl esters of those.All of these new and old compounds are lla-hydroxy compounds. Alsoincluded in Formula XVI are the corresponding but novel 11 fl-hydroxycompounds which are also encompassed by Formula V and which are usefulfor the pharmacological purposes described above either as such ortransformed into salts or esters as described above.

When one of these Formula-XVI compounds has the R or epi configurationfor the hydroxy at C-15, and the corresponding compound with the Sconfiguration at C-15 is desired, or when'one of these Formula-XVIcompounds has the S configuration for the hydroxy at C-15, and thecorresponding compound with the R or epi configuration at C-15 isdesired, those desired com pounds are made by the processes of Chart C.In Chart C, R R R and B, and are as defined above.

The overall process scheme of Chart C is to start with one particularC-l5 isomer of a compound encompassed by Formula XVI, i.e., either 15(8)or 15 (R). The C-15 hydroxy of that isomer is oxidized to a ketoniccarbonyl (XVII). Then, after an optional silylation of the C-9 and C-11hydroxy groups (XVIII), the C-15 carbonyl is reduced back to a secondaryhydroxy group. That reduction produces two C-15 hydroxy isomers, onewith S configuration and one with R or epi configuration. After removalof any silyl groups, the isomers XIX and XX are separated. One of theisomers will be the same compound used as starting material (XVI). Theother isomer will be the desired product. The starting material isomeris recycled to produce more of the desired isomer. This reaction schemehas previously been used to transform PGF to '15/3-PGF See Pike et al.,J. Org. Chem. 34, 3552 (1969).

CHART O \/V\/\/ H i; XVI

1(oxidation) ooon \/Y\/\/ H xvrr l (silylatlon) \/\O(\/\/ xvnI l(reduction) 1 hydrolysis L oon.

xxx

Referring now to Chart C, starting material XVI (from Chart B) is asingle compound, a mixture of two compounds, one with alpha and one withbeta configuration at C-9, or a mixture of four compounds, i.e.,9a,].1a, 9a,11fi, 9fl,11oc, and 913,115.

For the oxidation of XVI to XVII, any oxidizing agent can be used whichwill oxidize an allylic alcohol to an c p-unsaturated :ketone oraldehyde. Examples of those are2,3-dichloro-5,6-dicyano-1,4-benzoquinone, activated manganese dioxide,or nickel peroxide (see Fieser et al., Reagents for Organic Syntheses,John Wiley & Sons, Inc., New York, N.Y., 1967, pp. 215, 637, and 731).Alternatively, these oxidations are carried out by oxygenation in thepresence of the 15-hydroxyprostaglandin dehydrogenase of swine lung (seeArkiv for Kemi 25, 293 (1966)). These reagents are used according toprocedures known in the art. See, for example, J. Biol. Chem. 239, 4097(1964).

Regarding the transformation of XVII to XVIII in Chart C, it will beobserved that the parameters for XVIII are such that all XVII compoundsare included in XVIII. In other words, the transformation of XVII toXVIII is an optional process step in proceeding from XVII to XIX and XX.The reason for this is as follows. During the reduction of XVIII to XIXand XX, the ratio of XIX to XX obtained will be diiferent when R inXVIII is hydrogen than when R is Si-(A) For example, reduction of theFormula-XVIII 9a,11u-isomer wherein R and R are both hydrogen with zincborohydride gives the corresponding Formula-XIX and -XX in the amounts43 parts of XIX (R or epi configuration) and 57 parts of XX (Sconfiguration). On the other hand, when R, in the Formula-XVIII reactantis 'Si(A) the amounts with the same reducing agent are 27 parts of XIXand 73 parts of XX. Similar differences are observed with the otherisomers encompassed by Formula XVIII although not necessarily in thesame direction. Accordingly, whether R in Formula XVIII is to behydrogen or Si-(A) depends on the particular C-15 isomer desired and theinfiuenee of silylation n the isomer ratio. For any particularFormula-XVII starting material, the latter is readily determined bysmall scale reductions with and without silylation. When silylation:before carbonyl reduction is indicated, largely for economic reasons,it is preferred that A be methyl, i.e., that R, be (CH --Si-.

These silylations are carried out as described above for the Chart A andChart B silylation.

The carbonyl reduction of XVIII to XIX is carried out as described abovefor the transformation of PGE-type Formula-XIV compounds to PGF-typeFormula-XV compounds. As for those reductions, the sodium, potassium andzinc borohydrides are preferred as reducing agents, especially zincborohydride.

When the method used to isolate the carbonyl reduction product does notremove any Si-(A) groups which may be present, that is accomplished asdescribed above for the removal of -Si-(A) groups from Formula-X IIproducts (Chart A, XII to XIII). q

The Formula-XIX and -XX products are separated from each other bymethods known in the art, for example, silica gel chromatography. See,for example, Pike et al., J. Org. Chem. 34, 3552 (196 9) for this typeof separation.

If one of the isomers or isomer mixtures of Formulas XIX or XX is notdesired for a pharmacological use as such or transformed to esters orpharmacologically acceptable salts as described above, that isomer orisomer mixture is recycled as a Formula-XVI starting material in theprocesses of Chart C to produce additional 0f the desired isomer.

The products of Formulas XIX and XX wherein the configuration of theC-11 hydroxy is beta are encompassed by Formula V. The products of[Formula XIX wherein the configuration of the C-11 hydroxy is alpha areencompassed by Formula VII. The intermediates of Formula XVII areencompassed by Formula VI. Thus, all of the compounds are useful for thepharmacological purposes described above for the Formula V, VI and VIIcompounds. The compounds prepared as in Chart C are also useful to makethe other esters and the pharmacologically acceptable salts of theFormula V, VI, and VI I compounds also as described above.

There are two particular embodiments of the novel process of thisinvention which are especially preferred. One of those embodimentsprovides an optional route to PGF and starts with lSB-PGA acetate methylester, the most abundant component of Plexaura homomalla (Esper), 1792,forma -R. The other embodiment provides a preferred route of PGE andstarts with PGA readily obtained as described above by maintainingcolonies or colony pieces of Plexaum homomalla (Esper), 1792, forma S incontact with water in a temperature range up to 50 C. untilsubstantially free of PGA 15 acetate methyl ester.

The first of these embodiments is shown in Chart D, and the second isshown in Chart E. All of these Chart D and Chart -E reactions andreagents for effecting them are CHART D 1513-1 GA: acetate methyl ester1519-1 GFzq 15-aeetate methyl ester (saponification) -1 GFZn 1(oxidation) 15-oxo-P GFM:

l (silylation) 15-oxo-P GFh 9,11-di-Si-(A); ether l (reduction,hydrolysis) 155-? GFia and PGF2 I (an l{/sepraon) inseam.

CHART n P GA,

l (silylation) PGA; 15-Si-(A); ether l (oxidation) P GA; 15-Si-(A);other a and B 10, 11-epoxides 1 (reduction, hydrolysis) P GE; and 11 8-1GE, l (separation) PGE;

described generically and specifically above, and all are exemplifiedbelow. In Charts D and B, it is preferred that Si(A) be Si(CH Also inCharts D and B, it is optional whether silylation of 15-oxo-PGF (ChartD) or PGA (Chart E) produces the corresponding -Si--(A) ester-ether oronly the ether.

The invention is more fully understood by the following examples andpreparations:

All temperatures are in degrees centigrade.

Ultraviolet spectra are recorded on a Cary Model 15 spectrophotometer.

The collection of chromatographic eluate fractions starts when theeluant front reaches the bottom of the column.

Brine, herein, refers to an aqueous saturated sodium chloride solution.

The A-IX solvent system used in thin layer chromatography (TLC) is madeup from ethyl acetate-acetic acid- 2,2,4 trimethylpentane-water(90:20:50z100) according to M. Hamberg and B. Samuelsson, J. Biol. Chem.241, 257 (1966).

PREPARATION 1 To distinguish Plexaura homomalla (Esper), 1792, forma Rfrom Plexaura homomalla (Esper), 1792, forma S, a TLC method is used. Aspecimen approximately 2 cm. in length is harvested and placed in asmall vial, with a small amount of water if necessary to insure it iswet, and kept closed for 6-24 hrs. About one ml. of methanol is thenadded and the sample is either shaken for 2 hrs. at about 25 C. or isstored for 16-24 hrs. at about 10 C. A sample of the liquid (10-21 isspotted on a TLC plate. It is preferred to use a fluorescent-treatedsilica gel plate, e.g. Um'plate Silica Gel GF (Analtech, Inc., Newark,Del.). As reference standards, spots of PGA-,, and /3-PGA are alsoapplied. The plate is developed in the A-IX system (Hamberg andSamuelsson, J. Biol. Chem. 241, 257 (1965) The spots are finallyvisualized with vanillin-phosphoric acid spray (McAleer, Arch. Biochem.E. Biophys. 66, 120 (1957)). Comparison of the unknown with the tworeference spots is then made and the identity of the coral established(forma S corresponding to PGA forma R to 15fl-PGA PREPARATION 2 PGA fromPlexaura homomalla (Eper), 1792 forma S Colonies of Plexaura homomalla(Esper), 1792, forma S, collected from reefs oil? the north shore ofJamaica, are frozen by contact with solid carbon dioxide within one hourafter removal from the reef waters. The frozen colonies are maintainedin insulated boxes containing solid carbon dioxide (temperature belowabout C.) until ready for thawing. Then, the frozen colonies (700 g.)are ground to a small particle size (Waring Blendor) and mixed with 1500ml. of water. The mixture is maintained about 20 hrs. at about C. withstirring. Then, the mixture is filtered through a pad of diatomaceousearth, and the filtrate is acidified with con centrated hydrochloricacid to pH about 2-3. The acidified filtrate is extracted four timeswith ethyl acetate. The extracts are combined, filtered, washed withbrine, dried with anhydrous sodium sulfate, and evaporated under reducedpressure to give 11 g. of oily residue.

The solid residue on the diatomaceous earth filter pad is stirred 2hours in methanol (enough to cover said residue) at 25 C. The mixture isthen filtered, and the filtrate is evaporated to give 14 g. of oilyresidue.

The two oily residues are combined and chromatographed on 1500 g. ofacid-washed silica gel, eluting successively with 8 l. of a 25 to 65%gradient of ethyl acetate in Skellysolve B, 8 1. of a 65 to gradient ofethyl acetate in Skellysolve B, and 5 l. of 2% methanol in ethylacetate, collecting 500 ml. fractions. (Skellysolve B is a mixture ofisomeric hexanes.) Fractions 8-12 are combined and evaporated to give asmall amount of PGA containing a trace of PGA methyl ester. Fractions15-18 are combined and evaporated to give 9.54 g. of PGA Fractions 35-40are combined to give 0.414 g. of PGE2.

PREPARATION 3 1519-PGA from Plexaura Homomalla (Esper), 1792, forma RColonies of Plexaura homomalla (Esper), 1792, forma R, collected fromreefs off the southeast shore of Florida near Miami, are chopped intochunks several inches long. The chunks are frozen by contact with solidcarbon dioxide with one hour after removal from the reef waters. Thefrozen colony pieces are maintained in insulated boxes containing solidcarbon dioxide (temperature below about -20 C.) until ready for thawing.Then, colony pieces (600 g.) are mixed with 1500 ml. of water. Themixture is stirred and maintained at 25 C. for 23 hours. The mixture isthen filtered through a pad of diatomaceous earth, and the filtrate isacidified to pH about 2-3 with concentrated hydrochloric acid. Theacidified filtrate is extracted four times with ethyl acetate. Theextracts are combined, filtered, washed with brine, dried with anhydroussodium sulfate, and evaporated to give 9.2 g. of oily residue.

The solid residue on the diatomaceous earth pad is stirred 15 hours inmethanol (enough to cover said residue) at 25 C. The mixture is thenfiltered, and the filtrate is evaporated. The residue is dissolved inethyl acetate, and the solution washed successively with 3 Nhydrochloric acid and brine, dried with anhydrous sodium sulfate, andevaporated to give 5.83 g. of an oily residue.

The second oily residue and 8.2 g. of the first oily residue arecombined and chromatographed on one kg. of acid-washed silica gel,eluting successively with 3-1. portions of 25%, 35%, 45%, 55%, and 65%ethyl acetate in Skellysolve B, collecting 500-ml. fractions. Fractions18-22 are combined and evaporated to give 5.54 g. of ISfi-PGA Fractions15-17 are combined and evaporated to give 1.37 g. of 15,8-PGA methylester.

PREPARATION 4 PGA; compounds from Plexaura homomalla (Esper), 1792,forma S Frozen colonies of Plexaurw homomalla (Esper), 1792, forma S(see Preparation 2) are broken manually into pieces several cm. inlength. The pieces 500 g.) are then covered with methanol and themixture is maintained for 3 hours at 25 C. The mixture is then ground ina Waring blender and filtered, and the filtrate is evaporated underreduced pressure. The residue is dissolved in ethyl acetate, and thesolution is washed successively with one N hydrochloric acid, water, andbrine, dried with anhydrous sodium sulfate, and evaporated under reducedpressure.

The oily residue is chromatographed on 2 kg. of acidwashed silica gelwet-packed with Skellysolve B (a mixture of isomeric hexanes), elutingwith 24 1. of a 25 to 100% ethyl acetate in Skellysolve B gradient. Thefractions which contain PGA acetate methyl ester, PGA acetate, PGAmethyl ester, and PGA as shown by TLC with the A-IX system areseparately combined and evaporated to give those compounds.

PREPARATION 15B-PGA compounds from Plexaura homomalla (Esper), 1792,forma R Colonies of Plexaura homomalla (Esper), 1792, forma R, collectedfrom reefs off the southeast shore of Florida near Miami, are choppedinto chunks several inches long. The chunks are frozen by contact withsolid carbon dioxide within one hour after removal from the reef waters.The frozen colony pieces are maintained in insulated boxes containingsolid carbon dioxide (temperature below about 20 C.) until the time forextraction. Then, the frozen colony pieces are ground to a smallparticle size (Mitts and Merrill Hogger; average largest dimension about5 mm.). The particles (1500 g.) are then stirred at high speed with 5gallons of dichloromethane for 20 minutes at about 25 C. externaltemperature. The mixture of dichloromethane and particles is thenfiltered through a pad of diatomaceous earth, and the filtrate isevaporated to about a 2-liter volume at 30 C. under reduced pressure.The liquid which remains is Washed with water, dried with sodiumsulfate, and evaporated at 30 C. under reduced pressure.

The oily residue (60 g.) is chromatographed on 3 kg. of silica gel wetpacked in Skellysolve B (a mixture of isomeric hexanes), elutingsuccessively with a gradient of 4 l. of Skellysolve B and 4 l. of 20%ethyl acetate in Skellysolve B, 27 l. of 20%, 18 l. of 50%, and 8 l. of75% ethyl acetate in Skellysolve B, collecting 600-ml. fractions.Fractions 39-60 are combined and evaporated to give 24.3 g. of ISB-PGAacetate methyl ester. Between fractions 60 and 74 those fractions shownby TLC to contain ISfl-PGA acetate are combined and evap orated to yieldthat compound. Fractions 74-76 are combined and evaporated to give 1.03g. of 15B-PGA methyl ester. Fractions 83-91 are combined and evaporatedto give 1.08 g. of 15B-PGE 15-acetate methyl ester. Still laterfractions shown by TLC to contain l5/3-PGE methyl ester are combined andevaporated to yield that com-' pound.

Detection of the respective compounds by TLC is done by methods known inthe art, e.g. by spotting the extract fractions of a TLC silica gelplate alongside spots of the authentic compounds, developing the platewith A-IX system, and observing which spots of the extract fractionscorrespond exactly to the spots of the authentic compounds.

Following the procedures of Preparation 5, but substituting Plexaurahomomalla Esper), 1792, forma S for the Plexaura homomalla (Esper),1792, forma R of that example, there are obtained the correspondingcompounds of 15 (S) configuration, viz.: PGA acetate methyl ester, PGAacetate, PGA methyl ester, PGE 15-acetate methyl ester, and PGE methylester.

PREPARATION 6 PGA and 5,6-trans-PGA Separation of PGA;, from5,6-trans-PGA is done on a chromatographic column using asilver-saturated ion exchange resin. Preferably a macroreticular ionexchange resin is used, e.g. a sulfonated styrene-divinylbenzenecopolymer having surface area of 40-50 sq. m./g., 3040% porosity, andtotal exchange capacity of 4.5-5.0 meq. per gram of dry resin, forexample Amberlyst 15, available from Rohm and Haas Co., Philadelphia,Pa. The acidform resin is packed in a column, washed with warm water,and converted to the silver form by passing a 10% silver nitratesolution through the column until the efiluent shows a pH of 3.5-4.0.The column is then Washed with water to remove ionic silver, and finallywith denatured ethanol (Type 3A). A solution of a mixture of PGA, and5,6-trans-PGA e.g. fractions 15-18 of Preparation 2, in ethanol ischarged to the column. Elution with 3A alcohol then yields fractionswhich are combined according to their content of 5,6-trans-PGA(faster-eluting) or PGA Testing for the presence of 5,6-trans-PGA or PGAin the eluate is conveniently done by TLC using silver nitrate-treatedsilica gel plates (e.g. Analtech Uniplates dipped in saturated ethanolicsilver nitrate and dried) and developing with the A-IX system. R, of PGAis 0.45; R, of 5,6-trans-PGA is 0.50. Combined fractions areconcentrated, partitioned between dichloromethane and a little water,dried over sodium sulfate, and concentrated under reduced pressure toyield the title compounds.

For quantitatively assaying the 5,6-trans-PGA content of mixtures of PGAand 5,6-trans-PGA a combination thin-layer-spectrophotometric assay isused. Silica gel-impregnated glass microfiber sheets (e.g. I'I'LC sheetsof the Gelman Instrument Co., Ann Arbor, Mich.) are impregnated withsilver nitrate, using 5% ethanolic silver nitrate and drying. Spots of100 to 200 ,ug. of the PGA mixture are applied and developed in thesolvent system 2,2,4-trimethylpentane:ethyl acetateracetic acidzwater(100:35: 8: l0, uper phase). The sheet is dried and sprayed withRhodamine 6G (Applied Science Co., State College, Pa.) and viewed underultraviolet light. The areas containing the cis and trans materials (R;of PGA =0.6; R; of 5,6-trans-PGA =0.7) are marked, then excised andeluted with methanol (1.9 ml.) and potassium hydroxide solution (0.1 ml.of 45%). After incubation at 40 for 30 min., the respective solutionsare centrifuged and analyzed spectrophotometrically at 278 nm.

Following the procedure of Preparation 6, 5,6-trans- ISB-PGA isseparated from 15 fi-PGA PREPARATION 7 PGE and 5,6-trans-PGE Followingthe procedure of Preparation 6, PGE is separated from 5,6-trans-P-GE asfollows. A solution of a mixture of PGE and 5,6-trans- PGE is charged tothe column. Elution with 3A alcohol yields fractions which are combinedaccording to their content of 5 ,6-trans-PGE (faster eluting) or PGEAssay for 5,-6-trans-PGE or PGE is done by TLC as for the PGA -typecompounds above. Rf of PGE is 0.13; R, of 5,6-trans-PGE is 0.17.Combined fractions are concentrated, dried over sodium sulfate, andconcentrated under reduced pressure to yield the title compounds.

PREPARATION 8 PGA 15-acetate methyl ester, separation from 5,6-trans-PGA, 15-acetate methyl ester A mixture of PGA; 15-acetate methyl esterand 5,6- trans-PGA 15-acetate methyl ester (11.0 g., :15) is dissolvedin 415 ml. of a solution of methanol-wateracetic acid (-5-04) andmercuric acetate (6.1 g.), and left standing at about 25 C. for 30 min.Water (250 ml.) is added and the mixture extracted twice with 700 ml. ofSkellysolve B. The Skellysolve .B phase is washed with ml. of 60%methanol, dried over sodium sulfate, and concentrated to an oil (4.35g.) having a high content of 5,6- trans-PGA 15-acetate methyl ester. Theaqueous methanol phase is acidified with 32 ml. of 6 N hydrochloric acidand the mixture is extracted with two portions of 700 ml. of SkellysolveB. The organic phase is dried over sodium sulfate and concentrated to anoil (5.53 g.). This last material is subjected to the same proceduresagain, using 350 ml. of the methanol-water-acetic acid and 4.6 g. ofmercuric acetate. There is recovered from the work-up 31 of theaqueous-methanol phase a fraction (3.92 g.) of the title compoundcontaining only a small percentage of the 5,6-trans-PGA compound.

Following the procedure of Preparation 8, 5,6-trans- 15,8-PGA 15-acetatemethyl ester is separated from 15,3- PGA IS-acetate methyl ester.

In the following examples, the above-described 5,6- trans-PG and5,6-trans-15 3-PG compounds are subjected to the same transformations asthe P6 and 15,8-PG compounds disclosed herein and illustrated hereafter.

EXAMPLE 1 15,6-PGA methyl ester A solution of 70% aqueous perchloricacid (50 drops) in 50 drops of water is added to a solution of ISB-PGAacetate methyl ester (2.0 g.) in 100 ml. of methanol. The mixture isstirred for 15 hours at 25 C. and then diluted with 80 ml. of water. Themethanol is removed under reduced pressure, and the aqueous residue isextracted with ethyl acetate. The extract is washed successively withwater and brine, dried with anhydrous sodium sulfate, and evaporated.The residue is chromatographed on 200 g. of silica gel, eluting with 2.5l. of a gradient of 2070% ethyl acetate in Skellysolve B (a mixture ofisomeric hexanes), collecting 100-ml. fractions. Fractions 15-19 arecombined and evaporated to give 727 mg. of 15fi-PGA methyl ester.

EXAMPLE 2 PGA methyl ester A solution of lfi-PGA methyl ester (250 mg.)in 20 ml. of anhydrous tetrahydrofuran is cooled to 0 C. in anatmosphere of nitrogen. Tributylamine (1.5 ml.) is added, and themixture is stirred at 0 C. while adding methanesulfonyl chloride (1 ml.)dropwise. The mixture is stirred 30 minutes at 0 C. Then, 10 ml. ofwater is added, and the mixture is allowed to warm to 25 C. and isstirred for one hour. The tetrahydrofuran is evaporated under reducedpressure, and the aqueous residue is extracted with ethyl acetate. Theextract is washed successively with one N hydrochloric acid, water, andbrine, dried with anhydrous sodium sulfate, and evaporated. The residueis chromatographed on 30 g. of silica gel, eluting with 800 ml. of agradient of -70% ethyl acetate in Skellysolve B, collecting -ml.fractions. Fractions 14-16 are combined and evaporated to give 58 mg. ofPGA, methyl ester. Fractions 12 and 13 are combined to give 49 mg. ofthe starting material, ISfi-PGA methyl ester.

Following the procedure of Example 2, PGA methyl ester is transformed toa mixture of PGAg and 15 3-PGA methyl esters, the two compounds beingseparated as in Example 2.

EXAMPLE 3 'PGA 15-formate and 15fl-PGA 15-formate A solution of sodiumcarbonate (50 mg.) in 7.5 ml. of anhydrous'formic acid is added to PGA(0.25 g.). This mixture is stirred under nitrogen at 25 C. for 2 hrs.The reaction mixture is concentrated under reduced pressure, taken up inbenzene, and again concentrated under reduced pressure. The residue ischromatographed on acid- Washed silica gel (e.g. Mallinckrodt SilicarCC-4), eluting with a gradient of 25-75 ethyl acetate-Skellysolve B(isomeric hexane mixture) and collecting fractions. Those fractionsshown by TLC to contain the respective 15- formate compound, separatedfrom its isomer and free of starting material and impurities, arecombined and concentrated under reduced pressure to give the titlecompounds.

EXAMPLE 4 PGA and 15 ,8-PGA PGA 15-formate (100 mg., Example 3) isdissolved in a mixture of 10 ml. of methanol and 2.5 ml. of saturatedaqueous sodium bicarbonate solution. The solution is stirred undernitrogen at 25 C. for 2.5 hrs. Then 5 ml. of water and 2 ml. of 1 Nhydrochloric acid are added, and the solution concentrated. The aqueousresidue is adjusted to pH 23 and extracted three times with ethylacetate. The combined extracts are washed with water, dried over sodiumsulfate, and concentrated to yield PG'A Similarly, hydrolysis of ISB-PGA15-formate (Example 3) yields 15, 3-PGA EXAMPLE 5 l5/3-PGA 10,11-epoxideacetate methyl ester Hydrogen peroxide (350 ml.; 30% aqueous) is addedwith stirring to a solution of ISB-PGA acetate methyl ester (265 g.) in5000 ml. of methanol under a nitrogen atmosphere at 20 C. Then, one Naqueous potassium hydroxide solution (50 ml.) is added gradually duringone hour with stirring at 20 C. The mixture is stirred an additional 2hours at --20 C. Then, one N hydrochloric acid ml.) is added, and themethanol is removed under reduced pressure at 35 C. The residue isdissolved in 300 ml. of ethyl acetate, and the solution is washed 3times with 500-ml. portions of Water. The combined water washes areextracted with 300 ml. of ethyl acetate. The two ethyl acetate solutionsare combined, washed with brine, dried with anhydrous sodium sulfate andevaporated to give 2.75 g. of a mixture of the alpha and beta10,11-epoxides of 15,8-PGA acetate methyl ester.

EXAMPLE 6 PGA 10,11-epoxide methyl ester Hydrogen peroxide (0.3 ml.; 30%aqueous) and one N aqueous sodium hydroxide (0.5 ml.) are added to asolution of PGA methyl ester (229 mg.) in 10 ml. of isopropyl alcohol at0 C. After 2.5 hours at 0 C., 10 ml. of water and one ml. of one :Nhydrochloric acid are added, and the isopropyl alcohol is removed underreduced pressure. The residue is extracted with ethyl acetate. Theextract is washed successively with water and brine, dried withanhydrous sodium sulfate, and evaporated. The residue is chromatographedon 30 g. of silica gel, eluting with 800 ml. of a gradient of 20-70%ethyl acetate in Skellysolve B, collecting 25-ml. fractions. Fractions5-10 are combined and evaporated to give 136 mg. of a mixture of thealpha and beta 10,11-epoxides of PGA methyl ester.

EXAMPLE 7 PGA acetate methyl ester or and S 10,11-epoxides Refer toChart A.

A solution of PGA IS-acetate methyl ester (1.954 g.) in 30 ml. ofdimethoxyethane (DME) is cooled to 55 C. under nitrogen, and 5.25 ml. oft-butyl hydroperoxide is added. Then, 5 ml. of 0.25 N methanolic lithiumhydroxide (prepared from the mono-hydrate) is added over min. Afterabout 46 hrs. an additional 2.5 ml. of the base is added over 50 min.Finally, after about 23.5 hrs. the reaction is complete, as shown byTLC. The mixture is acidified to pH 5-6 with 1 N hydrochloric acid andis concentrated under reduced pressure. The residue is taken up in ethylacetate, washed with brine, dried over sodium sulfate, and concentratedunder reduced pressure. The product, 2.0 g., contains the title alphaand beta compounds in a ratio 015 621, respectively, as shown by gaschromatography.

Following the procedures of Example 7, but replacing the lithiumhydroxide solution with methanolic magnesium methoxide (prepared frommagnesium and anhydrous methanol), there is obtained a productcontaining the alpha and beta epoxides in a ratio of 4: 1.

Following the procedures of Example 7, but replacing the DME with amixture of toluene-DME (10:1) and replacing the lithium hydroxide withTriton B (benzyltrimethylammonium hydroxide) in methanol, there areobtained the alpha and beta epoxides in a ratio of 7.2:1.

Following the procedures of Example 7, but replacing the DME with amixture of toluene-DME (1:1) and holding the reaction temperature at--40 C., the product contains the alpha and beta epoxides in a ratio of6.2:1.

Following the procedures of Example 7, but replacing the DME withtetrahydrofuran (TI-IF) and replacing the lithium hydroxide solutionwith Triton B, there are obtained the alpha and beta epoxides in a ratioof 4.5: 1.

EXAMPLE 8 15,8-PGE 15-acetate methyl ester and 11,18,155-PGE 15-acetatemethyl ester Granular aluminum metal (50 g.) is added to a solution ofmercuric chloride (50 g.) in 2 l. of Water. The mixture is swirled untilhydrogen gas evolution starts to become vigorous (about 30 seconds).Then, most of the aqueous solution is decanted, and the rest is removedby rapid filtration. The amalgamated aluminum is washed rapidly andsuccessively with two ZOO-ml. portions of methanol and two ZOO-ml.portions of anhydrous diethyl ether. The amalgamated aluminum is thencovered with anhydrous diethyl ether until used.

Methanol (250 ml.) and water (25 ml.) are added to a solution of amixture of the alpha and beta 10,11- epoxides of 15B,PGA acetate methylester (275 g.) in 2500 ml. of diethyl ether. The mixture is cooled to'-10 C. and the amalgamated aluminum prepared as above from 50 g. ofaluminum metal is added. The mixture is stirred and maintained at about25 C. with external cooling. After one hour, amalgamated aluminumprepared as above from 50 g. of aluminum metal is added. After anadditional hour of stirring at 25 C., amalgamated aluminum prepared asabove from 50 g. of aluminum metal and also 25 ml. of water are added.After an additional hour of stirring at 25 C., 100 g. of magnesiumsulfate is added as a filter aid, and the mixture is filtered. Thefilter cake is Washed thoroughly with dichloromethane, and the combinedfiltrate and washings are evaporated at 25 C. under reduced pressure togive a mixture (247 g.) of 15;?- PGE IS-acetate methyl ester and11,8,15fi-PGE 15-acetate methyl ester.

Part of this mixture (210 g.) is chromatographed on 30 kg. of silica gelwet-packed with 60 l. of 25% ethyl acetate in Skellysolve B (6-inchdiameter column), eluting successively with 60-1. portions of 25%, 30%,35%, 40%, 45%, 50%, 55%, and 60% ethyl acetate in Skellysolve B,collecting 4-1. fractions. Fractions 71-76 are combined and evaporatedto give 27 g. of 1118,155-PGE l5-acetate methyl ester. Fractions 81-98are combined and evaporated to give 115 g. of ISB-PGE 15-acetate methylester.

EXAMPLE 9 l5fl-PGE l5-acetate methyl ester and 11fl,l5,B-PGE 15-acetatemethyl ester Anhydrous sodium acetate (0.5 g.) and zinc dust (500 mg.)are added to a solution of a mixture of the alpha and beta epoxide of15fi-PGA acetate methyl ester, prepared as in Example 5, in 5 ml. ofacetic acid. This mixture is stirred at 25 C. in an atmosphere ofnitrogen and cooled to about 15 C. One-half ml. of a solution ofchromium (III) chloride hexahydrate (300 mg.) in 1 ml. of water isadded, and the mixture is stirred at 0 C. for 3 hours. The mixture isthen diluted with ethyl acetate, and the solution is washed successivelywith four portions of water, one N hydrochloric acid, sodium bicarbonatesolution, and brine, dried with anhydrous sodium sulfate, andevaporated. The residue is chromatographed on 20 g. of silica gel,eluting with 600 ml. of a gradient of 20-75% ethyl acetate inSkellysolve B, collecting 25-ml. fractions. Fractions 10 and 11 arecombined to give 115,1518-PGE 15-acetate methyl ester. Fractions 13 and14 are combined to give ISB-PGE IS-acetate methyl ester.

EXAMPLE 10 PGE methyl ester and llfl-PGE methyl ester Freshly preparedchromium (l1) acetate (450 mg., argon atmosphere; Inorganic Syntheses,8, is added to a solution of 136 mg. of epoxides (Example 6) in amixture of 3 ml. of acetic acid and one ml. of water in an atmosphere ofargon at 0 C. The mixture is stirred at 5 C. under argon for 18 hours.Ice is then added to the mixture, and that mixture is extracted withethyl acetate. The extract is washed successively with water, one Nhydrochloric acid, sodium bicarbonate solution, and brine, dried withanhydrous sodium sulfate, and evaporated. The residue is chromatographedon silica gel (20 g.), eluting with 600 ml. of a gradient of 20-100%ethyl acetate in Skellysolve B, collecting 20 ml. fractions. Fractions19- 22 are combined and evaporated to give 27 mg. of 11/3- PGE methylester. Fractions 24-27 are combined and evaporated to give 5 mg. of PGEmethyl ester.

EXAMPLE 11 PGE and llfl-PGE Hydrogen peroxide (0.35 ml.; 30% aqueous) isadded to a solution of PGA (200 mg.) in 5 ml. of methanol. The mixtureis cooled to 20 C., and 0.75 ml. of one N aqueous sodium hydroxidesolution is slowly added with stirring. After one hour of stirring at 200., one ml. of one N hydrochloric acid is added, and the mixture isevaporated under reduced pressure. The residue is dissolved in ethylacetate, and the resulting solution is washed successively with waterand brine, dried with anhydrous sodium sulfate, and evaporated. Theresidue is treated with amalgamated aluminum as described in Example 8,using 2.5 ml. diethyl ether, 0.25 ml. methanol, and 0.03 ml. water, theamalgamated aluminum being added in 2 portions. When the reduction iscomplete, ethyl acetate and one N hydrochloric acid are added to thereaction mixture and the mixture is separated in a separatory funnel.The ethyl acetate layer is washed successively with one N hydrochloricacid, water, and brine, dried with anhydrous sodium sulfate, andevaporated. The residue is subjected to preparative thin layerchromatography to give PGE and 11,6-PGE in the ratio 1:2.

EXAMPLE 12 15 3-PGE and 11fl,15,8-PGE Following the procedure of Example11, 15,3-PGA is transformed to 15,8-PGE and 11,8,15fi-PGE those beingobtained in the ratio 1:1.

EXAMPLE 13 PGEZ and Hexamethyldisilizane (1 ml.) andtn'methylchlorosilane (0.2 ml.) are added with stirring to a solution ofPGA (250 mg.) in 4 ml. of tetrahydrofuran at 0 C. under nitogen. Thismixture is maintained at 5 C. for 15 hours. The mixture is thenevaporated under reduced pressure. Toluene is added and evaporatedtwice. Then the residue is dissolved in 6 ml. of methanol, and thesolution is cooled to --20 C. Hydrogen peroxide (0.45 ml.; 30% aqueous)is added. Then, one N sodium hydroxide solution (0.9 ml.) is addeddropwise with stirring at -20 C.

After 2 hours at -'-20 C., an additional 0.3 ml. of the sodium hydroxidesolution is added with stirring at -20 C. After another hour in therange -10 to -20 C., an additional 0.1 ml. of the sodium hydroxidesolution is added. Then, 1.5 ml. of one N hydrochloric acid is added,and the mixture is evaporated under reduced pressure. The residue isextracted with ethyl acetate, and the extract is Washed successivelywith one N hydrochloric acid brine, dried with anhydrous sodium sulfateand evaporated. The

residue is dissolved in ml. of diethyl ether. To this solution is added0.5 ml. of methanol and 0.1 ml. of water. Then, amalgamated aluminummade from 0.5 g. of aluminum metal as described in Example 8 is added insmall portions during 3 hours at 25 C. Then, ethyl acetate and 3 Nhydrochloric acid are added, and the ethyl acetate layer is separatedand washed successively with one N hydrochloric acid and brine, driedwith anhydrous sodium sulfate, and evaporated. The residue ischromatographed on 50 g. of acid-washed silica gel, eluting first with400 m1. of a gradient of 50100% ethyl acetate in Skellysolve B, and thenwith 100 ml. of 5% methanol in ethyl acetate, collecting 25 ml.fractions. Fractions 9 and are combined and evaporated to give 18 mg. ofIIp-PGE Fractions 17-25 are combined and evaporated to give 39 mg. ofPGE Following the procedures of Example 13 but replacing PGA with PGAacetate and optionally omitting the silylation step, there are obtainedthe corresponding PGE -acetate and llfi-PGE 15-acetate compounds.

EXAMPLE l4.-PGE

Refer to Chart C.

(a) Si1ylation.--A mixture of PGA (0.68 g.), 4 ml. of tetrahydrofuran(THF), and 1 ml. of trimethylchlorosilane solution (5% inhexamethyldisilazane is stirred under nitrogen for 2 hrs. at about 25 C.Then the silylated material is concentrated by removal of THF underreduced pressure, utilizing added benzene (10 ml.) to facilitate removalof THF.

(b) Oxidation.A cold (40 C.) solution of the above silylated material in15 ml. of isopropyl alcohol is mixed with 1.2 ml. of 30% aqueoushydrogen peroxide, followed by 1.5 ml. of 3 N aqueous lithium hydroxideadded dropwise. The temperature is allowed to warm to about 30 C. Thereaction is continued until the PGA has been exhausted as shown by theabsence of PGA in a thin layer chromatographic (TLC) spot test using theA-IX system (Hamberg and Samuelsson, J. Biol. Chem., 257 (1966). At 30'C., the reaction time is about 3-4 hrs. After completion 5 ml. of 1 Nhydrochloric acid is added and the mixture is concentrated under reducedpressure. The residue is extracted with ethyl acetate, washed with 0.5 Nhydrochloric acid and then brine, dried over sodium sulfate, andconcentrated to the epoxide.

(c) Reduction and hydrolysis.--A solution of the above epoxide in ml. ofTHF and 2 ml. of methanol is stirred with 4 ml. of saturated aqueoussodium bicarbonate solution and cooled to 15 C. To the mixture is added,in portions with vigorous stirring, an aluminum amalgam made from 1 g.of powdered aluminum (Example *8). After stirring at about C. for 45min., a sample is analyzed by TLC for PGE and epoxide. Reaction iscontinued if necessary. When the epoxide is no longer present, thesupernatant suspension is decanted from the aluminum which is furtherwashed with ethyl acetate. The combined decantate and washes areconcentrated under reduced pressure. The residue is taken up in about15-20 ml. of ethyl acetate and shaken with 20 ml. of 1 N hydrochloricacid. The layers are separated, the organic phase is washed with 0.5 Nhydrochloric acid,

1 and then brine, dried and concentrated to an oily residue of 0.837 g.

(d) Separation.A solution of the above residue in a small amount of 20%ethyl acetate-Skellysolve B (isomeric hexanes) is applied to achromatographic column of 65 g. of acid-washed silica, e.g. MallinckrodtSilicar CC-4. Elution with a gradient of 20-100% ethyl acetate-Skellysolve B gives fractions. Those fractions which are shown by TLC tocontain the desired compound are combined, and concentrated. There isobtained in separate fractions PGE 0.5 g., and 1lfl-PGE 0.05 g.

Alternatively, the oily product from 0 above is triturated in ethylacetate-cyclohexane (1:1), cooled to about 10 C. and seeded to yieldcrystalline PGE about 0.4 g.

36 The mother liquor is subjected to silica gel chromatography to yieldseparate fractions of about 0.1 g. PGE and 0.05 g. llfl-PGE EXAMPLE 15.PGE

(a) Silylation.'Following the procedure of Example 14, step a, PGA (0.68g.) is silylated and concentrated.

(b) Oxidation.-A cold (40 C.) solution of the above silylated materialin 3.0 ml. of isopropyl alcohol is mixed with 0.84 ml. of t-butylhydroperoxide, followed by the addition of 0.352 g. of lithium hydroxidemonohydrate. The reaction is continued at -40 C. to 20 C. for 5 hrs.Then, the pH of the mixture is adjusted to about 8.0 by adding carbondioxide.

(c) Reduction and hydrolysis-The above mixture is diluted with 20 ml. ofTHF and cooled to 15 C. To the mixture is added in portions withvigorous stirring, an aluminum amalgam made from 1 g. of powderedaluminum (Example 8). After stirring at about 25 C. for 45 min., asample is analyzed by TLC for PGE and epoxide. Reaction is continued ifnecessary. When the epoxide is no longer present, the supernatantsuspension is decanted from the aluminum which is further washed withethyl acetate. The combined decantate and Washes are concentrated underreduced pressure. The residue is taken up in about 15-20 ml. of ethylacetate and shaken with 20 ml. of 1 N hydrochloric acid. The layers areseparated, the organic phase is washed with 0.5 N hydrochloric acid, andthen brine, dried and concentrated to an oily residue of 0.837 g.

(d) Separation.A solution of the above residue in a small amount of 20%ethyl acetate-Skellysolve B (isomeric hexanes) is applied to achromatographic column of 65 g. of acid-washed silica, e.g. MallinckrodtSilicar CC-4. Elution with a gradient of 20-100% ethyl acetate-Skellysolve B gives fractions. Those fractions which are shown by TLC tocontain the desired compound are combined, and concentrated. There isobtained in separate fractions PGE 0.5 g., and llfi-PGE 0.05 g.

EXAMPLE 16 15B-PGE 11-tSi-(CH ether 15-acetate methyl esterHexamethyldisilazane g.) and then trimethylchlorosilane (20 g.) areadded to a solution of 15,8-PGE 15-acetate methyl ester (Example 8) in400 ml. of tetrahydrofuran with vigorous stirring at 25 C. undernitrogen. The reaction mixture is maintained in the range 20 to 25 C. byexternal cooling, and is stirred 2 hours under nitrogen. Then, themixture is evaporated at 50 C. at reduced pressure. The residue is mixedwith ml. of toluene, and the mixture is filtered through a pad ofdiatomaceous earth. The filtrate is evaporated at 50 C. under reducedpressure. The residue is mixed with 150 ml. of toluene, and again thetoluene is removed under reduced pressure at 50 C. to give 75 g. of -PGE11-Si-(CH ether 15acetate methyl ester.

Following the procedure of Example 16, 1119-15 3- PGE 15-acetate methylester is transformed to the corresponding 1l-Si-(CH ether.

. Also following the procedure of Example 16, but using larger amountsof hexamethyldisilazane and trimethylchlorosilane, 15B-PGE methyl ester,ISB-PGE 116,155- PGE methyl ester, and 1l;8,15B-PGE are transformed tothe corresponding 11,15-di-Si(CH ethers.

EXAMPLE l7 15 8-PGF l5-acetate methyl ester and IS S-PGF 15-acetatemethyl ester Sodium borohydride (1.42 g.) is added in one portion to asolution of 15fl-PGE 1t1-Si-(CH ether IS-acetate methyl ester (Example16) (30.7 g.) in 500 ml. of absolute ethanol at 0 C. with stirring. Themixture is stirred at 0 C. for 3.5 hours. Then, 10 ml. of glacial aceticacid is added slowly with stirring at 0 C. Then,

100 ml. of water is added, and the mixture is allowed to warm to 25 C.with stirring, and is stirred 15 hours at 25 C. The ethanol isevaporated under reduced pressure, and the residue is mixed with 400 ml.of brine. The mixture is extracted with 3 portions of ethyl acetate (400ml., 250 ml., and 150 ml.). The combined extracts are washedsuccessively with two 100-ml. portions of water, 100 ml. of saturatedaqueous sodium bicarbonate solution, two 100 ml. portions of brine,dried with anhydrous sodium sulfate, and evaporated under reducedpressure to give 24.5 g. of a mixture of ISB-PGF 15- acetate methylester and ISB-PGF IS-acetate methyl ester.

Aqueous sodium hydroxide solution (10%; 275 ml.) is added to a solutionof 48 g. of a mixture of 15;t-PGF 15-acctate methyl ester and 15,8-PGFIS-acetate methyl ester (Example 17) in 350 m1. of methanol at C. withstirring under nitrogen. The mixture is allowed to warm to 25 C. withstirring, and is stirred 3 hours at 25 C. Then, the methanol isevaporated under reduced pressure at 35 C. The aqueous residue is cooledand extracted once with a mixture of diethyl ether and dichloromethane1:1). Then, the aqueous residue is acidified with 260 ml. of 3 Nhydrochloric acid, saturated with sodium chloride, and extracted with 3portions of ethyl acetate (400 ml., 250 ml., and 150 ml.). The combinedextracts are washed successively with two 100-m1. portions of water andtwo 100-ml. portions of brine, dried with anhydrous sodium sulfate, andevaporated to give 42 g. of a mixture of 15fl-PGF and l5B-PGF EXAMPLE 1915-Oxo-PGF and 15-Oxo-PGF The mixture of 15fi-PGF and lifi-PGF- (42 g.)obtained as in Example 18 is dissolved in 950 ml. of dioxane. To thissolution at 25 C. is added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (40g.). This mixture is stirred 18 hours at 50 C. under nitrogen. Themixture is then cooled to 25 C. and filtered. The filter cake is washedwith dichloromethane, and the combined filtrate and washing areevaporated under reduced pressure at 45 C. to give a mixture (66 g.) of15-oxo-PGF and 15-oxo-PGF Part of this mixture (33 g.) ischromatographed on 3 kg. of acid-washed silica gel, eluting successivelywith l. 60%, 10 1. 70%, 101. 80%, 20 l. 90% ethyl acetate in SkellysolveB, l. ethyl acetate, and 10 1. 5% methanol in ethyl acetate, collecting650-ml. fractions. Fractions 42-53 are combined and evaporated to give8.3 g. of 15- oxo-PGF Fractions 64-85 are combined and evaporated togive 3.3 g. of 15-oxo-PGF EXAMPLE PGF2, and ISB-PGFZ,

Hexamethyldisilazane (70 m1.) and trimethylchlorosilane (14 ml.) areadded with vigorous stirring to a solution of 15-oxo-PGF (3.0 g.) in 350ml. of tetrahydrofuran at C. under nitrogen. The mixture is stirred 18hours at 25 C. under nitrogen. Then, the mixture is evaporated underreduced pressure at 50 C. Toluene (100 ml.) is added to the residue, andthe mixture is filtered through a pad of diatomaceous earth. Thefiltrate is evaporated, and 100 ml. of toluene is added to the residue.This mixture is evaporated under reduced pressure to give the9,11-di-Si-(CH ether of 15-oxo-PGF This disilyl ether is dissolved in 20ml. of 1,2-dimethoxyethane. Sodium borohydride (680 mg.) is suspended in65 ml. of 1,2-dimethoxyethane at 0 C. under nitrogen. Anhydrous zincchloride (1.23 g.) is added to this suspension, and the mixture isstirred minutes at 0 C. Then, the solution of the disilyl ether is addeddropwise during 10 minutes with stirring at 0 C. The resulting mixtureis allowed to warm to 25 C. with stirring, and is stirred 4 hours at 25C. Then, 30 ml. of water is added, followed by 8 ml. of glacial aceticacid. This mixture is stirred 15 hours at 25 C. The mixture is thenpoured into a mixture of ice and 100 ml. of 0.5 N hydrochloric acid.That mixture is saturated with sodium chloride, and then extracted withseveral portions of ethyl acetate. The combined extracts are washed withbrine dried with anhydrous sodium sulfate, and evaporated under reducedpressure. The residue (3.2 g.) is chromatographed on 600 g. ofacid-washed silica gel, eluting successively with 5 l. of ethyl acetatein Skellysolve B, 5 l. of ethyl acetate in Skellysolve B, and 5 l. of agradient of 90% ethyl acetate and 10% methanol in ethyl acetate,collecting 550ml. fractions. Fractions 21-26 are combined and evaporatedto give 543 mg. of 15fl-PGF Fractions 28-36 are combined and evaporatedto give 1.62 g. of PGF EXAMPLE 21 15fl-PGF 15-acetate methyl ester andISB-PGF IS-acetate methyl ester Sodium borohydride (6.0 g.) is added inone portion with vigorous stirring to a solution of 15,8-PGE 11-Si- (CHether, lS-acetate methyl ester (77 g.) in 1500 ml. of methanol at 30 C.Then 5.0 g. sodium borohydride is added and the mixture is stirred onehour at 30 C., and a second hour while warming to 20 C. Then, glacialacetic acid (30 ml.) is added slowly, followed by ml. of water. Thismixture is stirred 15 hours at 25 C., and then evaporated under reducedpressure. The residue is mixed with brine (2 volumes), and the mixtureis extracted 3 times with ethyl acetate. The combined extracts arewashed successively with water, sodium bicarbonate solution, and brine,dried with anhydrous sodium sulfate, and evaporated under reducedpressure to give 60 g. of a mixture of 15fi-PGF 15-acetate methyl esterand lSfi-PGF 15-acetate methyl ester.

Part (21 g.) of this mixture is chromatographed on 1.8 kg. of silicagel, wet-packed in 50% ethyl acetate in Skellysolve B, elutingsuccessively with 20. l. 50%, 20 l. 60%, and 5 1. 75% ethyl acetate inSkellysolve B, collecting 650-m1. fractions. Fractions 12-26 arecombined and evaporated to give 22.0 g. of 15 8-PGF 15-acetate methylester. Fractions 44-61 are combined and evaporated to give 6.11 g. of15fi-PGF 15-acetate methyl ester.

EXAMPLE 22.15,B-PGF Following the procedure of Example 18, 15,8-PGF 15-acetate methyl ester is saponified to 15p-PGF EXAMPLE 23.1513PGFFollowing the procedure of Example 18, ISB-PGF IS-acetate methyl esteris saponified to 15p-PGF EXAMPLE 24.-15-Oxo-PGF Following the procedureof Example 19, l5l3-PGF is oxidized to 15-oxo-PGF EXAMPLE 25.15-Oxo-PGFFollowing the procedure of Example 19, IS S-P61 is oxidized to15-oxo-PGF EXAMPLE 26.-PGF and 15,3-PGF Following the procedure ofExample 20, 15-oxo-PGF is silylated and then reduced to a mixture of PGFand ISB-PGF which are separated as described for the alpha compounds inExample 20.

EXAMPLE 27.1l}3,l5B-PGF and 1118,1513-PGF Following the procedures ofExamples 16 and 21 11/3, ISB-PGE 15-acetate methyl ester is transformedto a mixture of IIBJSB-P GF 15-acetate methyl ester and 39 115,15fl-PGFl-acetate methyl ester which are separated as described for the productsof Example 21. Those acetate methyl esters are then separatelysaponified as in Example 18 to give 1118,15fl-PGF and 11p,15p-PGFFollowing the procedure of Example 19, 115,1518-PGF is oxidized to11,8-15-oxo-PGF Following the procedure of Example 19, 11,19,15fl-PGF isoxidized to 11}8,15-oxo-PGF Following the procedure of Example 20,l1B-15-oxo- PGF is silylated and then reduced to a mixture of 111+ PGFand 115,155-PGF which are separated as described for the products ofExample 20.

EXAMPLE 3l.-l1/3-PGF and 11 [-3-15B-PGF Following the procedure ofExample 20, 11fi-15-ox0- PGF is silylated and then reduced to a mixtureof 11B- PGF and 115,15B-PGF which are separated as described for theproducts of Example 20.

EXAMPLE 32.-PGF and PGF (a) Silylation.-Following the procedure ofExample 14, step a PGA (0.68 g.) is silylated and concentrated.

(b) Oxidation.A cold (-40 C.) solution of the above silylated materialin 15 ml. of isopropyl alcohol is mixed with 1.2 ml. of 30% aqueousperoxide, followed by 1.5 ml. of 3 N aqueous lithium hydroxide addeddropwise. The reaction is continued at about 30 C. for 4 hrs. Then, thepH of the mixture is adjusted to about 8.0 by adding carbon dioxide andthe mixture is concentrated under reduced pressure.

(c) Reduction and hydrolysis-The residue above is taken up in 20 ml. ofTHF and 2 ml. of methanol. To it is added with vigorous stirring, analuminum amalgam made from 1 g. of powdered aluminum (Example 8). Afterthe epoxide is no longer present as shown by TLC, the mixture is heatedto -60 C. for about one hour to reduce all 9-oxo compounds to 9-hydroxycompounds. There is thereby formed a mixture containing P P lip-P and115-1 salts. The mixture is cooled and the supernatant material isdecanted from the aluminum which is further washed with ethyl acetate.The combined decantate and washes are concentrated under reducedpressure. The residue is taken up in ethyl acetate and acidified, whilestirring, with 20 ml. of l N hydrochloric acid. The layers are separatedand the organic phase is Washed with 0.5 N hydrochloric acid and thenbrine dried and concentrated under reduced pressure.

(d) Separation.-A solution of the above residue in a small amount of 20%ethyl acetate-Skellysolve B (isomeric hexanes) is applied to achromatographic column of acid-washed silica, e.g. Mallinckrodt SilicarCC-4. Elution with a gradient of 20-100% ethyl acetate-Skellysolve Bgives fractions. Those fractions which are shown by TLC to contain PGFare combined and concentrated to yield PGF Likewise, those fractionsshown to contain PGF 11/9-PGF or 11 3-PGF are combined and concentratedto yield those compounds, respectively.

EXAMPLE 33.-PGF and PGFgp Hydrogen peroxide (3.5 ml., 30% aqueous) isadded with stirring to a solution of PGA; acetate methyl ester (2.65 g.)in 50 ml. of methanol under a nitrogen atmosphere at -20 C. Then, 5.0ml. of 0.1 N aqueous potassium hydroxide solution is added graduallyover one hr. with stirring at -20 C. The mixture is stirred anadditional2 hrs. at -20 C. Then, carbon dioxide is added to the mixture to adjustthe pH to about 7.0-8.0, and the mixture containing the alpha and beta10,11-epox ides is concentrated under reduced pressure.

The above residue is taken up in ml. of THF and 8 ml. of methanol, andcooled to l0 C. To the mixture is added in portions, with vigorousstirring, an aluminum amalgam made from 4 g. of powdered aluminum(Example 8). The mixture is stirred and maintained at about 25 C. withcooling. After an hour, a sample is analyzed by TLC for 10,1l-epoxide ofPGA l5-acetate methyl ester and for PGE IS-acetate methyl ester.Reaction is continued at about 25 C. until substantially no epoxide isleft. The reaction mixture is then warmed to 60 C., with stirring. Afterone hour a sample is analyzed by TLC for PGE; 15-acetate methyl esterand 11(3- PGE 15-acetate methyl ester and for the corresponding PGF andPGF compounds. Reaction is continued at 60 C. until no PGE: compoundsare left. The mixture is cooled and the supernatant material is decantedfrom the aluminum which is further washed with ethyl acetate. Thecombined decantate and washes are concentrated under reduced pressure.The residue is taken up in ethyl acetate and chromatographed on silicagel, eluting with 20l00% ethyl acetate-Skellysolve B, collectingfractions. Those fractions which are shown by TLC to contain PGF15-acetate methyl ester are combined and concentrated to give thatcompound. Likewise, the fractions containing PGF 15-acetate methyl esterare combined and concentrated to give that compound.

PGF and PGF- are obtained by saponification of the above corresponding15-acetate methyl esters. Aqueous sodium hydroxide (2.75 ml. 10%) isadded to a solution of PGF IS-acetate methyl ester (0.48 g.) in 10 ml.of methanol at 0 C. with stirring under nitrogen. The mixture is allowedto warm to 25 C. with stirring and is stirred 3 hrs. at 25 C. Themixture is partially concentrated under reducedpressure. The aqueousresidue is cooled and extracted once with a mixture of diethyl ether anddichloromethane (1:1). The aqueous residue is aciditied with about 2.6ml. of 3 N hydrochloric acid, saturated with sodium chloride, andextracted with ethyl acetate. The organic phase is washed with water andbrine, dried with anhydrous sodium sulfate, and concentrated to PGF In asimilar fashion, PGF is obtained by saponification of PGF IS-acetatemethyl ester.

I claim:

1. A process for producing an alpha epoxide of the formula whereinrepresents the alpha configuration, wherein R is hydrogen or methyl, andwherein G is wherein R is hydrogen, acetyl, or -Si(A) wherein A is alkylof one to 4 carbon atoms, inclusive, aralkyl of 7 to 12 carbon atoms,inclusive, phenyl, or phenyl substituted with one or 2 fluoro, chloro,or alkyl of one to 4 carbon atoms, inclusive, which comprises startingwith a compound of the formula

